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Current Research and Scholarly Interests

Our goal is to elucidate the fundamental basis of gene regulation. We study the control of transcription, the first step in the pathway of gene expression. Current work focuses on discovery of the molecular machines involved in transcription, reconstitution of the process with purified components, structure determination of the transcription machinery, and structure-function relationships in chromatin, the natural DNA template for transcription.

Highlights of work from the past three years include:

1. Discovery of a human homolog of the 20-protein yeast Mediator complex. Mediator is the central processing unit of gene regulation, receiving both positive and negative inputs and transducing the information to the transcription machinery.

2. Structure determination of 10-subunit, half million Dalton RNA polymerase II, in the act of transcription, with template DNA and protduct RNA, by X-ray crystallography at atomic resolution.

Publications

All Publications

Abstract

Biochemical and structural studies have shown that the initiation of RNA polymerase II transcription proceeds in the following stages: assembly of the polymerase with general transcription factors and promoter DNA in a 'closed' preinitiation complex (PIC); unwinding of about 15 base pairs of the promoter DNA to form an 'open' complex; scanning downstream to a transcription start site; synthesis of a short transcript, thought to be about 10 nucleotides long; and promoter escape. Here we have assembled a 32-protein, 1.5-megadalton PIC derived from Saccharomyces cerevisiae, and observe subsequent initiation processes in real time with optical tweezers. Contrary to expectation, scanning driven by the transcription factor IIH involved the rapid opening of an extended transcription bubble, averaging 85 base pairs, accompanied by the synthesis of a transcript up to the entire length of the extended bubble, followed by promoter escape. PICs that failed to achieve promoter escape nevertheless formed open complexes and extended bubbles, which collapsed back to closed or open complexes, resulting in repeated futile scanning.

Abstract

Whereas RNA polymerase II (Pol II) transcription start sites (TSSs) occur about 30-35 bp downstream of the TATA box in metazoans, TSSs are located 40-120 bp downstream in S. cerevisiae. Promoter melting begins about 12 bp downstream in all eukaryotes, so Pol II is presumed to "scan" further downstream before starting transcription in yeast. Here we report that removal of the kinase complex TFIIK from TFIIH shifts the TSS in a yeast system upstream to the location observed in metazoans. Conversely, moving the normal TSS to an upstream location enables a high level of TFIIK-independent transcription in the yeast system. We distinguish two stages of the transcription initiation process: bubble formation by TFIIH, which fills the Pol II active center with single-stranded DNA, and subsequent scanning downstream, also driven by TFIIH, which requires displacement of the initial bubble. Omission of TFIIK uncouples the two stages of the process.

Abstract

Structure determination of gold nanoparticles (AuNPs) is necessary for understanding their physical and chemical properties, but only one AuNP larger than 1 nanometer in diameter [a 102-gold atom NP (Au102NP)] has been solved to atomic resolution. Whereas the Au102NP structure was determined by x-ray crystallography, other large AuNPs have proved refractory to this approach. Here, we report the structure determination of a Au68NP at atomic resolution by aberration-corrected transmission electron microscopy, performed with the use of a minimal electron dose, an approach that should prove applicable to metal NPs in general. The structure of the Au68NP was supported by small-angle x-ray scattering and by comparison of observed infrared absorption spectra with calculations by density functional theory.

Abstract

Recent evidence suggests that transcript elongation by RNA polymerase II (RNAPII) is regulated by mechanical cues affecting the entry into, and exit from, transcriptionally inactive states, including pausing and arrest. We present a single-molecule optical-trapping study of the interactions of RNAPII with transcription elongation factors TFIIS and TFIIF, which affect these processes. By monitoring the response of elongation complexes containing RNAPII and combinations of TFIIF and TFIIS to controlled mechanical loads, we find that both transcription factors are independently capable of restoring arrested RNAPII to productive elongation. TFIIS, in addition to its established role in promoting transcript cleavage, is found to relieve arrest by a second, cleavage-independent mechanism. TFIIF synergistically enhances some, but not all, of the activities of TFIIS. These studies also uncovered unexpected insights into the mechanisms underlying transient pauses. The direct visualization of pauses at near-base-pair resolution, together with the load dependence of the pause-entry phase, suggests that two distinct mechanisms may be at play: backtracking under forces that hinder transcription and a backtrack-independent activity under assisting loads. The measured pause lifetime distributions are inconsistent with prevailing views of backtracking as a purely diffusive process, suggesting instead that the extent of backtracking may be modulated by mechanisms intrinsic to RNAPII. Pauses triggered by inosine triphosphate misincorporation led to backtracking, even under assisting loads, and their lifetimes were reduced by TFIIS, particularly when aided by TFIIF. Overall, these experiments provide additional insights into how obstacles to transcription may be overcome by the concerted actions of multiple accessory factors.

Abstract

The protein density and arrangement of subunits of a complete, 32-protein, RNA polymerase II (pol II) transcription pre-initiation complex (PIC) were determined by means of cryogenic electron microscopy and a combination of chemical cross-linking and mass spectrometry. The PIC showed a marked division in two parts, one containing all the general transcription factors (GTFs) and the other pol II. Promoter DNA was associated only with the GTFs, suspended above the pol II cleft and not in contact with pol II. This structural principle of the PIC underlies its conversion to a transcriptionally active state; the PIC is poised for the formation of a transcription bubble and descent of the DNA into the pol II cleft.

Abstract

Whereas individual RNA polymerase II (pol II)-general transcription factor (GTF) complexes are unstable, an assembly of pol II with six GTFs and promoter DNA could be isolated in abundant homogeneous form. The resulting complete pol II transcription preinitiation complex (PIC) contained equimolar amounts of all 31 protein components. An intermediate in assembly, consisting of four GTFs and promoter DNA, could be isolated and supplemented with the remaining components for formation of the PIC. Nuclease digestion and psoralen cross-linking mapped the PIC between positions -70 and -9, centered on the TATA box. Addition of ATP to the PIC resulted in quantitative conversion to an open complex, which retained all 31 proteins, contrary to expectation from previous studies. Addition of the remaining NTPs resulted in run-off transcription, with an efficiency that was promoter-dependent and was as great as 17.5% with the promoters tested.

Abstract

A minimal RNA polymerase II (pol II) transcription system comprises the polymerase and five general transcription factors (GTFs) TFIIB, -D, -E, -F, and -H. The addition of Mediator enables a response to regulatory factors. The GTFs are required for promoter recognition and the initiation of transcription. Following initiation, pol II alone is capable of RNA transcript elongation and of proofreading. Structural studies reviewed here reveal roles of GTFs in the initiation process and shed light on the transcription elongation mechanism. This article is part of a Special Issue entitled: RNA Polymerase II Transcript Elongation.

Structure of the Mediator Head module bound to the carboxy-terminal domain of RNA polymerase IIPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICARobinson, P. J., Bushnell, D. A., Trnka, M. J., Burlingame, A. L., Kornberg, R. D.2012; 109 (44): 17931-17935

Abstract

The X-ray crystal structure of the Head module, one-third of the Mediator of transcriptional regulation, has been determined as a complex with the C-terminal domain (CTD) of RNA polymerase II. The structure reveals multiple points of interaction with an extended conformation of the CTD; it suggests a basis for regulation by phosphorylation of the CTD. Biochemical studies show a requirement for Mediator-CTD interaction for transcription.

Abstract

During transcription, RNA polymerase II (RNAPII) must select the correct nucleotide, catalyze its addition to the growing RNA transcript, and move stepwise along the DNA until a gene is fully transcribed. In all kingdoms of life, transcription must be finely tuned to ensure an appropriate balance between fidelity and speed. Here, we used an optical-trapping assay with high spatiotemporal resolution to probe directly the motion of individual RNAPII molecules as they pass through each of the enzymatic steps of transcript elongation. We report direct evidence that the RNAPII trigger loop, an evolutionarily conserved protein subdomain, serves as a master regulator of transcription, affecting each of the three main phases of elongation, namely: substrate selection, translocation, and catalysis. Global fits to the force-velocity relationships of RNAPII and its trigger loop mutants support a Brownian ratchet model for elongation, where the incoming NTP is able to bind in either the pre- or posttranslocated state, and movement between these two states is governed by the trigger loop. Comparison of the kinetics of pausing by WT and mutant RNAPII under conditions that promote base misincorporation indicate that the trigger loop governs fidelity in substrate selection and mismatch recognition, and thereby controls aspects of both transcriptional accuracy and rate.

Abstract

General transcription factor TFIIH, previously described as a 10-subunit complex, is essential for transcription and DNA repair. An eleventh subunit now identified, termed Tfb6, exhibits 45% sequence similarity to human nuclear mRNA export factor 5. Tfb6 dissociates from TFIIH as a heterodimer with the Ssl2 subunit, a DNA helicase that drives promoter melting for the initiation of transcription. Tfb6 does not, however, dissociate Ssl2 from TFIIH in the context of a fully assembled transcription preinitiation complex. Our findings suggest a dynamic state of Ssl2, allowing its engagement in multiple cellular processes.

Abstract

Structures of complete 10-subunit yeast TFIIH and of a nested set of subcomplexes, containing 5, 6, and 7 subunits, have been determined by electron microscopy (EM) and 3D reconstruction. Consistency among all the structures establishes the location of the "minimal core" subunits (Ssl1, Tfb1, Tfb2, Tfb4, and Tfb5), and additional densities can be specifically attributed to Rad3, Ssl2, and the TFIIK trimer. These results can be further interpreted by placement of previous X-ray structures into the additional densities to give a preliminary picture of the RNA polymerase II preinitiation complex. In this picture, the key catalytic components of TFIIH, the Ssl2 ATPase/helicase and the Kin28 protein kinase are in proximity to their targets, downstream promoter DNA and the RNA polymerase C-terminal domain.

Abstract

How does RNA polymerase recognize a promoter in duplex DNA? How are the DNA strands pried apart to enable RNA synthesis? A crystal structure by Feklistov and Darst unexpectedly reveals that these two processes are interconnected.

Abstract

Purified chromatin rings, excised from the PHO5 locus of Saccharomyces cerevisiae in transcriptionally repressed and activated states, were remodeled with RSC and ATP. Nucleosomes were translocated, and those originating on the promoter of repressed rings were removed, whereas those originating on the open reading frame (ORF) were retained. Treatment of the repressed rings with histone deacetylase diminished the removal of promoter nucleosomes. These findings point to a principle of promoter chromatin remodeling for transcription, namely that promoter specificity resides primarily in the nucleosomes rather than in the remodeling complex that acts upon them.

Abstract

The initiation of transcription by RNA polymerase II is a multistage process. X-ray crystal structures of transcription complexes containing short RNAs reveal three structural states: one with 2- and 3-nucleotide RNAs, in which only the 3'-end of the RNA is detectable; a second state with 4- and 5-nucleotide RNAs, with an RNA-DNA hybrid in a grossly distorted conformation; and a third state with RNAs of 6 nucleotides and longer, essentially the same as a stable elongating complex. The transition from the first to the second state correlates with a markedly reduced frequency of abortive initiation. The transition from the second to the third state correlates with partial "bubble collapse" and promoter escape. Polymerase structure is permissive for abortive initiation, thereby setting a lower limit on polymerase-promoter complex lifetime and allowing the dissociation of nonspecific complexes. Abortive initiation may be viewed as promoter proofreading, and the structural transitions as checkpoints for promoter control.

Abstract

Mediator is a key regulator of eukaryotic transcription, connecting activators and repressors bound to regulatory DNA elements with RNA polymerase II (Pol II). In the yeast Saccharomyces cerevisiae, Mediator comprises 25 subunits with a total mass of more than one megadalton (refs 5, 6) and is organized into three modules, called head, middle/arm and tail. Our understanding of Mediator assembly and its role in regulating transcription has been impeded so far by limited structural information. Here we report the crystal structure of the essential Mediator head module (seven subunits, with a mass of 223 kilodaltons) at a resolution of 4.3 ångströms. Our structure reveals three distinct domains, with the integrity of the complex centred on a bundle of ten helices from five different head subunits. An intricate pattern of interactions within this helical bundle ensures the stable assembly of the head subunits and provides the binding sites for general transcription factors and Pol II. Our structural and functional data suggest that the head module juxtaposes transcription factor IIH and the carboxy-terminal domain of the largest subunit of Pol II, thereby facilitating phosphorylation of the carboxy-terminal domain of Pol II. Our results reveal architectural principles underlying the role of Mediator in the regulation of gene expression.

Isolation of an activator-dependent, promoter-specific chromatin remodeling factorPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAEhrensberger, A. H., Kornberg, R. D.2011; 108 (25): 10115-10120

Abstract

Repressed PHO5 gene chromatin, isolated from yeast in the native state, was remodeled by yeast extract in a gene activator-dependent, ATP-dependent manner. The product of the reaction bore the hallmark of the process in vivo, the selective removal of promoter nucleosomes, without effect on open reading frame nucleosomes. Fractionation of the extract identified a single protein, chromodomain helicase DNA binding protein 1 (Chd1), capable of the remodeling activity. Deletion of the CHD1 gene in an isw1Δ pho80Δ strain abolished PHO5 gene expression, demonstrating the relevance of the remodeling reaction in vitro to the process in vivo.

Abstract

Optical absorption of a gold nanocluster of 102 Au atoms protected by 44 para-mercaptobenzoic acid (p-MBA) ligands is measured in the range of 0.05-6.2 eV (mid-IR to UV) by a combination of several techniques for purified samples in solid and solution phases. The results are compared to calculations for a model cluster Au(102)(SMe)(44) based on the time-dependent density functional theory in the linear-response regime and using the known structure of Au(102)(p-MBA)(44). The measured and calculated molar absorption coefficients in the NIR-vis region are comparable, within a factor of 2, in the absolute scale. Several characteristic features are observed in the absorption in the range of 1.5-3.5 eV. The onset of the electronic transitions in the mid-IR region is experimentally observed at 0.45 ± 0.05 eV which compares well with the lowest calculated transition at 0.55 eV. Vibrations in the ligand layer give rise to fingerprint IR features below the onset of low-energy metal-to-metal electronic transitions. Partial exchange of the p-MBA ligand to glutathione does not affect the onset of the electronic transitions, which indicates that the metal core of the cluster is not affected by the ligand exchange. The full spectroscopic characterization of the Au(102)(p-MBA)(44) reported here for the first time gives benchmarks for further studies of manipulation and functionalization of this nanocluster to various applications.

Abstract

The synthesis of Au(102)(p-MBA)(44) nanoparticles on a preparative scale in high yield is described. Various analytical methods are shown to give results consistent with the composition and known structure of the particles, showing the preparation is essentially homogeneous, and attesting to the validity of the methods as well. Derivatization of the particles with proteins and DNA is demonstrated, and conditions are described for imaging individual particles by cryo-EM at low electron dose, close to focus, conditions optimal for recording high-resolution details.

Abstract

A structurally conserved element, the trigger loop, has been suggested to play a key role in substrate selection and catalysis of RNA polymerase II (pol II) transcription elongation. Recently resolved X-ray structures showed that the trigger loop forms direct interactions with the beta-phosphate and base of the matched nucleotide triphosphate (NTP) through residues His1085 and Leu1081, respectively. In order to understand the role of these two critical residues in stabilizing active site conformation in the dynamic complex, we performed all-atom molecular dynamics simulations of the wild-type pol II elongation complex and its mutants in explicit solvent. In the wild-type complex, we found that the trigger loop is stabilized in the "closed" conformation, and His1085 forms a stable interaction with the NTP. Simulations of point mutations of His1085 are shown to affect this interaction; simulations of alternative protonation states, which are inaccessible through experiment, indicate that only the protonated form is able to stabilize the His1085-NTP interaction. Another trigger loop residue, Leu1081, stabilizes the incoming nucleotide position through interaction with the nucleotide base. Our simulations of this Leu mutant suggest a three-component mechanism for correctly positioning the incoming NTP in which (i) hydrophobic contact through Leu1081, (ii) base stacking, and (iii) base pairing work together to minimize the motion of the incoming NTP base. These results complement experimental observations and provide insight into the role of the trigger loop on transcription fidelity.

Mechanism of chromatin remodelingPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICALorch, Y., Maier-Davis, B., Kornberg, R. D.2010; 107 (8): 3458-3462

Abstract

Results from biochemical and structural studies of the RSC chromatin-remodeling complex prompt a proposal for the remodeling mechanism: RSC binding to the nucleosome releases the DNA from the histone surface and initiates DNA translocation (through one or a small number of DNA base pairs); ATP binding completes translocation, and ATP hydrolysis resets the system. Binding energy thus plays a central role in the remodeling process. RSC may disrupt histone-DNA contacts by affecting histone octamer conformation and through extensive interaction with the DNA. Bulging of the DNA from the octamer surface is possible, and twisting is unavoidable, but neither is the basis of remodeling.

Abstract

By adjustment of solvent conditions for synthesis, virtually monodisperse 4-mercaptobenzoic acid (p-MBA) monolayer-protected gold nanoparticles, 2 and 3 nm in diameter, were obtained. Large single crystals of the 2 nm particles could be grown from the reaction mixture. Uniformity was also demonstrated by the formation of two-dimensional arrays and by quantitative high-angle annular dark-field scanning transmission electron microscopy. The 2 and 3 nm particles were spontaneously reactive for conjugation with proteins and DNA, and further reaction could be prevented by repassivation with glutathione. Conjugates with antibody Fc fragment could be used to identify TAP-tagged proteins of interest in electron micrographs, through the binding of a pair of particles to the pair of protein A domains in the TAP tag.

Abstract

Previous x-ray crystal structures have given insight into the mechanism of transcription and the role of general transcription factors in the initiation of the process. A structure of an RNA polymerase II-general transcription factor TFIIB complex at 4.5 angstrom resolution revealed the amino-terminal region of TFIIB, including a loop termed the "B finger," reaching into the active center of the polymerase where it may interact with both DNA and RNA, but this structure showed little of the carboxyl-terminal region. A new crystal structure of the same complex at 3.8 angstrom resolution obtained under different solution conditions is complementary with the previous one, revealing the carboxyl-terminal region of TFIIB, located above the polymerase active center cleft, but showing none of the B finger. In the new structure, the linker between the amino- and carboxyl-terminal regions can also be seen, snaking down from above the cleft toward the active center. The two structures, taken together with others previously obtained, dispel long-standing mysteries of the transcription initiation process.

Abstract

The histone chaperone Vps75 forms a complex with, and stimulates the activity of, the histone acetyltransferase Rtt109. However, Vps75 can also be isolated on its own and might therefore possess Rtt109-independent functions. Analysis of epistatic miniarray profiles showed that VPS75 genetically interacts with factors involved in transcription regulation whereas RTT109 clusters with genes linked to DNA replication/repair. Additional genetic and biochemical experiments revealed a close relationship between Vps75 and RNA polymerase II. Furthermore, Vps75 is recruited to activated genes in an Rtt109-independent manner, and its genome-wide association with genes correlates with transcription rate. Expression microarray analysis identified a number of genes whose normal expression depends on VPS75. Interestingly, histone H2B dynamics at some of these genes are consistent with a role for Vps75 in histone H2A/H2B eviction/deposition during transcription. Indeed, reconstitution of nucleosome disassembly using the ATP-dependent chromatin remodeler Rsc and Vps75 revealed that these proteins can cooperate to remove H2A/H2B dimers from nucleosomes. These results indicate a role for Vps75 in nucleosome dynamics during transcription, and importantly, this function appears to be largely independent of Rtt109.

Schizosacharomyces pombe RNA polymerase II at 3.6-angstrom resolutionPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICASpahr, H., Calero, G., Bushnell, D. A., Kornberg, R. D.2009; 106 (23): 9185-9190

Abstract

The second structure of a eukaryotic RNA polymerase II so far determined, that of the enzyme from the fission yeast Schizosaccharomyces pombe, is reported here. Comparison with the previous structure of the enzyme from the budding yeast Saccharomyces cerevisiae reveals differences in regions implicated in start site selection and transcription factor interaction. These aspects of the transcription mechanism differ between S. pombe and S. cerevisiae, but are conserved between S. pombe and humans. Amino acid changes apparently responsible for the structural differences are also conserved between S. pombe and humans, suggesting that the S. pombe structure may be a good surrogate for that of the human enzyme.

Abstract

Transcribing RNA polymerases oscillate between three stable states, two of which, pre- and posttranslocated, were previously subjected to x-ray crystal structure determination. We report here the crystal structure of RNA polymerase II in the third state, the reverse translocated, or "backtracked" state. The defining feature of the backtracked structure is a binding site for the first backtracked nucleotide. This binding site is occupied in case of nucleotide misincorporation in the RNA or damage to the DNA, and is termed the "P" site because it supports proofreading. The predominant mechanism of proofreading is the excision of a dinucleotide in the presence of the elongation factor SII (TFIIS). Structure determination of a cocrystal with TFIIS reveals a rearrangement whereby cleavage of the RNA may take place.

Abstract

ATP-dependent chromatin-remodeling complexes, such as RSC, can reposition, evict or restructure nucleosomes. A structure of a RSC-nucleosome complex with a nucleosome determined by cryo-EM shows the nucleosome bound in a central RSC cavity. Extensive interaction of RSC with histones and DNA seems to destabilize the nucleosome and lead to an overall ATP-independent rearrangement of its structure. Nucleosomal DNA appears disordered and largely free to bulge out into solution as required for remodeling, but the structure of the RSC-nucleosome complex indicates that RSC is unlikely to displace the octamer from the nucleosome to which it is bound. Consideration of the RSC-nucleosome structure and published biochemical information suggests that ATP-dependent DNA translocation by RSC may result in the eviction of histone octamers from adjacent nucleosomes.

Abstract

Structural, biochemical, and genetic studies have led to proposals that a mobile element of multisubunit RNA polymerases, the Trigger Loop (TL), plays a critical role in catalysis and can be targeted by antibiotic inhibitors. Here we present evidence that the Saccharomyces cerevisiae RNA Polymerase II (Pol II) TL participates in substrate selection. Amino acid substitutions within the Pol II TL preferentially alter substrate usage and enzyme fidelity, as does inhibition of transcription by alpha-amanitin. Finally, substitution of His1085 in the TL specifically renders Pol II highly resistant to alpha-amanitin, indicating a functional interaction between His1085 and alpha-amanitin that is supported by rerefinement of an alpha-amanitin-Pol II crystal structure. We propose that alpha-amanitin-inhibited Pol II elongation, which is slow and exhibits reduced substrate selectivity, results from direct alpha-amanitin interference with the TL.

Abstract

The rate-limiting step of transcriptional activation in eukaryotes, and thus the critical point for gene regulation, is unknown. Combining biochemical analyses of the chromatin transition at the transcriptionally induced PHO5 promoter in yeast with modeling based on a small number of simple assumptions, we demonstrate that random removal and reformation of promoter nucleosomes can account for stochastic and kinetic properties of PHO5 expression. Our analysis suggests that the disassembly of promoter nucleosomes is rate limiting for PHO5 expression, and supports a model for the underlying mechanism of promoter chromatin remodeling, which appears to conserve a single nucleosome on the promoter at all times.

Abstract

A comprehensive survey of single amino-acid substitution mutations critical for RNA polymerase function published in Journal of Biology supports a proposed mechanism for polymerase action in which movement of the polymerase 'bridge helix' promotes transcriptional activity in cooperation with a critical substrate-interaction domain, the 'trigger loop'.

Abstract

Structural information on nanometer-sized gold particles has been limited, due in part to the problem of preparing homogeneous material. Here we report the crystallization and x-ray structure determination of a p-mercaptobenzoic acid (p-MBA)-protected gold nanoparticle, which comprises 102 gold atoms and 44 p-MBAs. The central gold atoms are packed in a Marks decahedron, surrounded by additional layers of gold atoms in unanticipated geometries. The p-MBAs interact not only with the gold but also with one another, forming a rigid surface layer. The particles are chiral, with the two enantiomers alternating in the crystal lattice. The discrete nature of the particle may be explained by the closing of a 58-electron shell.

Abstract

New structures of RNA polymerase II (pol II) transcribing complexes reveal a likely key to transcription. The trigger loop swings beneath a correct nucleoside triphosphate (NTP) in the nucleotide addition site, closing off the active center and forming an extensive network of interactions with the NTP base, sugar, phosphates, and additional pol II residues. A histidine side chain in the trigger loop, precisely positioned by these interactions, may literally "trigger" phosphodiester bond formation. Recognition and catalysis are thus coupled, ensuring the fidelity of transcription.

Abstract

Yeast Mediator proteins interacting with Med17(Srb4) have been expressed at a high level with the use of recombinant baculoviruses and recovered in homogeneous form as a seven subunit, 223 kDa complex. Electron microscopy and single-particle analysis identify this complex as the Mediator head module. The recombinant head module complements "headless" Mediator for the initiation of transcription in vitro. The module interacts with an RNA polymerase II-TFIIF complex, but not with the polymerase or TFIIF alone. This interaction is lost in the presence of a DNA template and associated RNA transcript, recapitulating the release of Mediator that occurs upon the initiation of transcription. Disruption of the head module in a temperature-sensitive mutant in vivo leads to the release of middle and tail modules from a transcriptionally active promoter. The head module evidently controls Mediator-RNA polymerase II and Mediator-promoter interactions.

Abstract

A general method of rigid, specific labeling of proteins with gold clusters has been devised. The method relies on the conjugation of a glutathione monolayer-protected gold cluster (MPC) with a single chain Fv antibody fragment (scFv), mutated to present an exposed cysteine residue. Efficient formation of a gold-thiolate bond between the MPC and scFv depends on activation of the gold cluster by chemical oxidation. Once formed, the MPC-scFv conjugate is treated with a reductant to quench cluster reactivity. The procedure has been performed with an MPC with an average Au(71) core and an scFv directed against a tetrameric protein, the influenza neuraminidase. A complex of the MPC-scFv conjugate with the neuraminidase was isolated, and the presence of four gold clusters was verified by cryoelectron microscopy.

Chromatin remodeling by nucleosome disassembly in vitroPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICALorch, Y., Maier-Davis, B., Kornberg, R. D.2006; 103 (9): 3090-3093

Abstract

The RSC chromatin-remodeling complex completely disassembles a nucleosome in the presence of the histone chaperone Nap1 and ATP. Disassembly occurs in a stepwise manner, with the removal of H2A/H2B dimers, followed by the rest of the histones and the release of naked DNA. RSC and related chromatin-remodeling complexes may be responsible for the removal of promoter nucleosomes during transcriptional activation in vivo.

Abstract

Others have shown that yeast strains bearing a ts mutation in the Srb4 subunit of Mediator cease transcription of all mRNA at the restrictive temperature, in a manner virtually indistinguishable from a strain bearing a ts mutation in the largest subunit of RNA polymerase II. We find that srb4ts Mediator is defective for the stimulation of basal RNA polymerase II transcription at the restrictive temperature in vitro. Taken together, these findings lead to the suggestion that Mediator is required for basal RNA polymerase II transcription in vivo. On this basis, Mediator is identified as a general transcription factor, comparable in importance to RNA polymerase II and other general factors for the initiation of transcription. The possibility that Mediator serves as an anti-inhibitor, opposing the effects of global negative regulators, is largely excluded.

Defined DNA/nanoparticle conjugatesPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAAckerson, C. J., Sykes, M. T., Kornberg, R. D.2005; 102 (38): 13383-13385

Abstract

Glutathione monolayer-protected gold clusters were reacted by place exchange with 19- or 20-residue thiolated oligonucleotides. The resulting DNA/nanoparticle conjugates could be separated on the basis of the number of bound oligonucleotides by gel electrophoresis and assembled with one another by DNA-DNA hybridization. This approach overcomes previous limitations of DNA/nanoparticle synthesis and yields conjugates that are precisely defined with respect to both gold and nucleic acid content.

Abstract

Water-soluble monolayer-protected gold clusters (MPCs) have been an object of investigation by many research groups since their first syntheses were reported in 1998 and 1999. The basic requirements for a ligand to form a monolayer protecting a gold cluster were established some time ago for alkanethiolate MPCs, but there has been no such information published for water-soluble MPCs. We identify 6 new ligands capable of forming water-soluble MPCs, as well as 22 water-soluble ligands that fail to form MPCs. Our findings contribute not only to the definition of the requirements for MPC formation but also to the variety of MPCs available for applications in chemistry and biology.

Abstract

Mediator was discovered because of its activity in a yeast RNA polymerase II (pol II) transcription system - it is needed for the system to respond to a transcriptional activator. Mediator is the central link in the enhancer-->activator-->Mediator-->pol II-->promoter pathway. The transduction of regulatory signals through this pathway is crucial for transcription of almost all pol II promoters in all eukaryote organisms.

Abstract

An RNA polymerase II promoter has been isolated in transcriptionally activated and repressed states. Topological and nuclease digestion analyses have revealed a dynamic equilibrium between nucleosome removal and reassembly upon transcriptional activation, and have further shown that nucleosomes are removed by eviction of histone octamers rather than by sliding. The promoter, once exposed, assembles with RNA polymerase II, general transcription factors, and Mediator in a approximately 3 MDa transcription initiation complex. X-ray crystallography has revealed the structure of RNA polymerase II, in the act of transcription, at atomic resolution. Extension of this analysis has shown how nucleotides undergo selection, polymerization, and eventual release from the transcribing complex. X-ray and electron crystallography have led to a picture of the entire transcription initiation complex, elucidating the mechanisms of promoter recognition, DNA unwinding, abortive initiation, and promoter escape.

Chromatin remodeling by DNA bending, not twistingPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICALorch, Y., Davis, B., Kornberg, R. D.2005; 102 (5): 1329-1332

Abstract

Single-stranded regions (gaps) in nucleosomal DNA interfere with action of the RSC chromatin-remodeling complex, monitored by exposure of restriction endonuclease cutting sites. Single-strand breaks (nicks) in the DNA, by contrast, have no effect. Gaps on one side of the cutting site are inhibitory, but gaps on the other side are not. A gap >100 bp from the cutting site is as effective as a gap <20 bp from the site. These findings suggest a remodeling process involving bending, but not twisting, of the DNA and further point to the propagation of a bent region (loop or bulge) from one end of the nucleosome to the other.

Diffusion of nucleoside triphosphates and role of the entry site to the RNA polymerase II active centerPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICABatada, N. N., Westover, K. D., Bushnell, D. A., Levitt, M., Kornberg, R. D.2004; 101 (50): 17361-17364

Abstract

Nucleoside triphosphates (NTPs) diffuse to the active center of RNA polymerase II through a funnel-shaped opening that narrows to a negatively charged pore. Computer simulation shows that the funnel and pore reduce the rate of diffusion by a factor of approximately 2 x 10(-7). The resulting limitation on the rate of RNA synthesis under conditions of low NTP concentration may be overcome by NTP binding to an entry site adjacent to the active center. Binding to the entry site greatly enhances the lifetime of an NTP in the active center region, and it prevents "backtracking" and the consequent occlusion of the active site.

Abstract

Binding of a ribonucleoside triphosphate to an RNA polymerase II transcribing complex, with base pairing to the template DNA, was revealed by X-ray crystallography. Binding of a mismatched nucleoside triphosphate was also detected, but in an adjacent site, inverted with respect to the correctly paired nucleotide. The results are consistent with a two-step mechanism of nucleotide selection, with initial binding to an entry (E) site beneath the active center in an inverted orientation, followed by rotation into the nucleotide addition (A) site for pairing with the template DNA. This mechanism is unrelated to that of single subunit RNA polymerases and so defines a new paradigm for the large, multisubunit enzymes. Additional findings from these studies include a third nucleotide binding site that may define the length of backtracked RNA; DNA double helix unwinding in advance of the polymerase active center; and extension of the diffraction limit of RNA polymerase II crystals to 2.3 A.

Abstract

Previous work demonstrated the removal of nucleosomes from the PHO5 promoter upon transcriptional activation in yeast. Removal could occur by nucleosome disassembly or by sliding of nucleosomes away from the promoter. We have now activated the PHO5 promoter on chromatin circles following excision from the chromosomal locus. Whereas sliding would conserve the number of nucleosomes on the circle, we found that the number was diminished, demonstrating chromatin remodeling by nucleosome disassembly.

Abstract

The structure of an RNA polymerase II-transcribing complex has been determined in the posttranslocation state, with a vacancy at the growing end of the RNA-DNA hybrid helix. At the opposite end of the hybrid helix, the RNA separates from the template DNA. This separation of nucleic acid strands is brought about by interaction with a set of proteins loops in a strand/loop network. Formation of the network must occur in the transition from abortive initiation to promoter escape.

Abstract

The structure of the general transcription factor IIB (TFIIB) in a complex with RNA polymerase II reveals three features crucial for transcription initiation: an N-terminal zinc ribbon domain of TFIIB that contacts the "dock" domain of the polymerase, near the path of RNA exit from a transcribing enzyme; a "finger" domain of TFIIB that is inserted into the polymerase active center; and a C-terminal domain, whose interaction with both the polymerase and with a TATA box-binding protein (TBP)-promoter DNA complex orients the DNA for unwinding and transcription. TFIIB stabilizes an early initiation complex, containing an incomplete RNA-DNA hybrid region. It may interact with the template strand, which sets the location of the transcription start site, and may interfere with RNA exit, which leads to abortive initiation or promoter escape. The trajectory of promoter DNA determined by the C-terminal domain of TFIIB traverses sites of interaction with TFIIE, TFIIF, and TFIIH, serving to define their roles in the transcription initiation process.

Abstract

Single-copy gene and promoter regions have been excised from yeast chromosomes and have been purified as chromatin by conventional and affinity methods. Promoter regions isolated in transcriptionally repressed and activated states maintain their characteristic chromatin structures. Gel filtration analysis establishes the uniformity of the transcriptionally activated state. Activator proteins interact in the manner anticipated from previous studies in vivo. This work opens the way to the direct study of specific gene regions of eukaryotic chromosomes in diverse functional and structural states.

Association of the mediator complex with enhancers of active genesPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAKuras, L., Borggrefe, T., Kornberg, R. D.2003; 100 (24): 13887-13891

Abstract

The multiprotein Mediator complex has been shown to interact with gene-specific regulatory proteins and RNA polymerase II in vitro. Here, we use chromatin immunoprecipitation to analyze the recruitment of Mediator to GAL genes of yeast in vivo. We find that Mediator associates exclusively with transcriptionally active and not inactive GAL genes. This association maps to the upstream activating sequence, rather than the core promoter, and is independent of RNA polymerase II, general transcription factors, and core promoter sequences. These findings support the idea of Mediator as a primary conduit of regulatory information from enhancers to promoters in eukaryotic cells.

Abstract

Tfb4 is identified as a subunit of the core complex of yeast RNA polymerase II general transcription factor IIH (TFIIH) by affinity purification, by peptide sequence analysis, and by expression of the entire complex in insect cells. Tfb3, previously identified as a component of the core complex, is shown instead to form a complex with cdk and cyclin subunits of TFIIH. This reassignment of subunits resolves a longstanding discrepancy between yeast and human TFIIH complexes.

Abstract

The structure of an RNA polymerase II/general transcription factor TFIIF complex was determined by cryo-electron microscopy and single particle analysis. Density due to TFIIF was not concentrated in one area but rather was widely distributed across the surface of the polymerase. The largest subunit of TFIIF interacted with the dissociable Rpb4/Rpb7 polymerase subunit complex and with the mobile "clamp." The distribution of the second largest subunit of TFIIF was very similar to that previously reported for the sigma subunit in the bacterial RNA polymerase holoenzyme, consisting of a series of globular domains extending along the polymerase active site cleft. This result indicates that the second TFIIF subunit is a true structural homolog of the bacterial sigma factor and reveals an important similarity of the transcription initiation mechanism between bacteria and eukaryotes. The structure of the RNAPII/TFIIF complex suggests a model for the organization of a minimal transcription initiation complex.

Complete, 12-subunit RNA polymerase II at 4.1-angstrom resolution: Implications for the initiation of transcriptionPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICABushnell, D. A., Kornberg, R. D.2003; 100 (12): 6969-6973

Abstract

The x-ray structure of complete RNA polymerase II from Saccharomyces cerevisiae has been determined, including a heterodimer of subunits Rpb4 and Rpb7 not present in previous "core" polymerase II structures. The heterodimer maintains the polymerase in the conformation of a transcribing complex, may bind RNA as it emerges from the enzyme, and is in a position to interact with general transcription factors and the Mediator of transcriptional regulation.

Abstract

It has long been known that promoter DNA is converted to a nuclease-sensitive state upon transcriptional activation. Recent findings have raised the possibility that this conversion reflects only a partial unfolding or other perturbation of nucleosomal structure, rather than the loss of nucleosomes. We report topological, sedimentation, nuclease digestion, and ChIP analyses, which demonstrate the complete unfolding of nucleosomes at the transcriptionally active PHO5 promoter of the yeast Saccharomyces cerevisiae. Although nucleosome loss occurs at all promoter sites, it is not complete at any of them, suggesting the existence of an equilibrium between the removal of nucleosomes and their reformation.

Abstract

The Srb8, -9, -10, and -11 proteins of yeast have been isolated as a discrete, stoichiometric complex. The isolated complex phosphorylates the C-terminal domain (CTD) of the largest subunit of RNA polymerase II at serines 2 and 5. In addition to the previously reported human homologs of Srb10 and 11, we have identified TRAP230/ARC240 and TRAP240/ARC250 as the human homologs of Srb8 and Srb9, showing the entire Srb8/9/10/11 complex is conserved from yeast to humans.

Structural analysis of the RSC chromatin-remodeling complexPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAAsturias, F. J., Chung, W. H., Kornberg, R. D., Lorch, Y.2002; 99 (21): 13477-13480

Abstract

Electron microscopy of the RSC chromatin-remodeling complex reveals a ring of protein densities around a central cavity. The size and shape of the cavity correspond closely to those of a nucleosome. Results of nuclease protection analysis are consistent with nucleosome binding in the cavity. Such binding could explain the ability of RSC to expose nucleosomal DNA in the presence of ATP without loss of associated histones.

Abstract

The holoenzyme formed by RNA polymerase II (RNAPII) and the Mediator complex is the target of transcriptional regulators in vivo. A three-dimensional structure of the yeast holoenzyme has been generated from electron microscopic images of single holoenzyme particles. Extensive changes in Mediator conformation required for interaction with RNAPII have been modeled by correlating the polymerase-bound and free Mediator structures. Determination of the precise orientation of the RNAPII in the holoenzyme indicates that Mediator contacts are centered on the RNAPII Rpb3/Rpb11 heterodimer, the eukaryotic homolog of the alpha(2) homodimer involved in transcription regulation in prokaryotes. Implications for the possible mechanism of transcription regulation by Mediator are discussed.

Structural basis of transcription: alpha-Amanitin-RNA polymerase II cocrystal at 2.8 A resolutionPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICABushnell, D. A., Cramer, P., Kornberg, R. D.2002; 99 (3): 1218-1222

Abstract

The structure of RNA polymerase II in a complex with the inhibitor alpha-amanitin has been determined by x-ray crystallography. The structure of the complex indicates the likely basis of inhibition and gives unexpected insight into the transcription mechanism.

A trithorax-group complex purified from Saccharomyces cerevisiae is required for methylation of histone H3PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICANagy, P. L., Griesenbeck, J., Kornberg, R. D., Cleary, M. L.2002; 99 (1): 90-94

Abstract

Histone methylation has emerged as an important mechanism for regulating the transcriptional accessibility of chromatin. Several methyltransferases have been shown to target histone amino-terminal tails and mark nucleosomes associated with either euchromatic or heterochromatic states. However, the biochemical machinery responsible for regulating histone methylation and integrating it with other cellular events has not been well characterized. We report here the purification, molecular identification, and genetic and biochemical characterization of the Set1 protein complex that is necessary for methylation of histone H3 at lysine residue 4 in Saccharomyces cerevisiae. The seven-member 363-kDa complex contains homologs of Drosophila melanogaster proteins Ash2 and Trithorax and Caenorhabditis elegans protein DPY-30, which are implicated in the maintenance of Hox gene expression and regulation of X chromosome dosage compensation, respectively. Mutations of Set1 protein comparable to those that disrupt developmental function of its Drosophila homolog Trithorax abrogate histone methylation in yeast. These studies suggest that epigenetic regulation of developmental and sex-specific gene expression are species-specific readouts for a common chromatin remodeling machinery associated mechanistically with histone methylation.

Abstract

TAP tags and dot blot analysis have been used to measure the amounts of RNA polymerase II transcription proteins in crude yeast extracts. The measurements showed comparable amounts of RNA polymerase II, TFIIE, and TFIIF, lower levels of TBP and TFIIB, and still lower levels of Mediator and TFIIH. These findings are consistent with the presumed roles of the transcription proteins, but do not support the idea of their recruitment in a single large complex to RNA polymerase II promoters. The approach employed here can be readily extended to quantitative analysis of the entire yeast proteome.

Abstract

A three-subunit Hap complex that interacts with the RNA polymerase II Elongator was isolated from yeast. Deletions of genes for two Hap subunits, HAP1 and HAP3, confer pGKL killer-insensitive and weak Elongator phenotypes. Preferential interaction of the Hap complex with free rather than RNA polymerase II-associated Elongator suggests a role in the regulation of Elongator activity.

Abstract

Seven purified proteins may be combined to reconstitute regulated, promoter-dependent RNA polymerase II transcription: five general transcription factors, Mediator, and RNA polymerase II. The entire system has been conserved across species from yeast to humans. The structure of RNA polymerase II, consisting of 10 polypeptides with a mass of about 500 kDa, has been determined at atomic resolution. On the basis of this structure, that of an actively transcribing RNA polymerase II complex has been determined as well.

Abstract

Structures of a 10-subunit yeast RNA polymerase II have been derived from two crystal forms at 2.8 and 3.1 angstrom resolution. Comparison of the structures reveals a division of the polymerase into four mobile modules, including a clamp, shown previously to swing over the active center. In the 2.8 angstrom structure, the clamp is in an open state, allowing entry of straight promoter DNA for the initiation of transcription. Three loops extending from the clamp may play roles in RNA unwinding and DNA rewinding during transcription. A 2.8 angstrom difference Fourier map reveals two metal ions at the active site, one persistently bound and the other possibly exchangeable during RNA synthesis. The results also provide evidence for RNA exit in the vicinity of the carboxyl-terminal repeat domain, coupling synthesis to RNA processing by enzymes bound to this domain.

Abstract

The crystal structure of RNA polymerase II in the act of transcription was determined at 3.3 A resolution. Duplex DNA is seen entering the main cleft of the enzyme and unwinding before the active site. Nine base pairs of DNA-RNA hybrid extend from the active center at nearly right angles to the entering DNA, with the 3' end of the RNA in the nucleotide addition site. The 3' end is positioned above a pore, through which nucleotides may enter and through which RNA may be extruded during back-tracking. The 5'-most residue of the RNA is close to the point of entry to an exit groove. Changes in protein structure between the transcribing complex and free enzyme include closure of a clamp over the DNA and RNA and ordering of a series of "switches" at the base of the clamp to create a binding site complementary to the DNA-RNA hybrid. Protein-nucleic acid contacts help explain DNA and RNA strand separation, the specificity of RNA synthesis, "abortive cycling" during transcription initiation, and RNA and DNA translocation during transcription elongation.

Abstract

A protocol for the incorporation of SeMet into yeast proteins is described. Incorporation at a level of about 50% suffices for the location of Se sites in an anomalous difference Fourier map of the 0.5 MDa yeast RNA polymerase II. This shows the utility of the approach as an aid in the model-building of large protein complexes.

Abstract

RSC and SWI/SNF chromatin-remodeling complexes were previously reported to generate a stably altered nucleosome. We now describe the formation of hybrids between nucleosomes of different sizes, showing that the stably altered structure is a noncovalent dimer. A basis for dimer formation is suggested by an effect of RSC on the supercoiling of closed, circular arrays of nucleosomes. The effect may be explained by the interaction of RSC with DNA at the ends of the nucleosome, which could lead to the release 60--80 bp or more from the ends. DNA released in this way may be trapped in the stable dimer or lead to alternative fates such as histone octamer transfer to another DNA or sliding along the same DNA molecule.

Abstract

Structures of yeast Mediator complex, of a related complex from mouse cells and of thyroid hormone receptor-associated protein complex from human cells have been determined by three-dimensional reconstruction from electron micrographs of single particles. All three complexes show a division in two parts, a "head" domain and a combined "middle-tail" domain. The head domains of the three complexes appear most similar and interact most closely with RNA polymerase II. The middle-tail domains show the greatest structural divergence and, in the case of the tail domain, may not interact with polymerase at all. Consistent with this structural divergence, analysis of a yeast Mediator mutant localizes subunits that are not conserved between yeast and mammalian cells to the tail domain. Biochemically defined Rgr1 and Srb4 modules of yeast Mediator are then assigned to the middle and head domains.

Abstract

Core TFIIH from yeast, made up of five subunits required both for RNA polymerase II transcription and nucleotide excision DNA repair, formed 2D crystals on charged lipid layers. Diffraction from electron micrographs of the crystals in negative stain extended to about 13 angstrom resolution, and 3D reconstruction revealed several discrete densities whose volumes corresponded well with those of individual TFIIH subunits. The structure is based on a ring of three subunits, Tfb1, Tfb2, and Tfb3, to which are appended several functional moieties: Rad3, bridged to Tfb1 by SsI1; SsI2, known to interact with Tfb2; and Kin28, known to interact with Tfb3.

Abstract

Three-dimensional reconstructions of icosahedral viruses from cryoelectron microscope images have reached resolutions where the microscope depth of field is a significant resolution-limiting factor. An analytical treatment presented here shows how the depth of field limitation can be understood as an envelope function which gradually attenuates the signal, starting well before the numerical depth of field is actually reached. A simple modification to the well-known back-projection reconstruction algorithm is described, called the defocus-gradient corrected back-projection, which computationally corrects for the contrast transfer function along a defocus gradient. Computer simulations demonstrate how the algorithm effectively eliminates the depth of field limitation.

Abstract

Mediator, a multiprotein complex involved in the regulation of RNA polymerase II transcription, binds to nucleosomes and acetylates histones. Three lines of evidence identify the Nut1 subunit of Mediator as responsible for the histone acetyltransferase (HAT) activity. An "in-gel" HAT assay reveals a single band of the appropriate size. Sequence alignment shows significant similarity of Nut1 to the GCN5-related N-acetyltransferase superfamily. Finally, recombinant Nut1 exhibits HAT activity in an in-gel assay.

Abstract

A backbone model of a 10-subunit yeast RNA polymerase II has been derived from x-ray diffraction data extending to 3 angstroms resolution. All 10 subunits exhibit a high degree of identity with the corresponding human proteins, and 9 of the 10 subunits are conserved among the three eukaryotic RNA polymerases I, II, and III. Notable features of the model include a pair of jaws, formed by subunits Rpb1, Rpb5, and Rpb9, that appear to grip DNA downstream of the active center. A clamp on the DNA nearer the active center, formed by Rpb1, Rpb2, and Rpb6, may be locked in the closed position by RNA, accounting for the great stability of transcribing complexes. A pore in the protein complex beneath the active center may allow entry of substrates for polymerization and exit of the transcript during proofreading and passage through pause sites in the DNA.

Abstract

A yeast strain harboring a temperature-sensitive allele of TFB3 (tfb3(ts)), the 38-kDa subunit of the RNA polymerase II transcription/nucleotide excision repair factor TFIIH, was found to be sensitive to ultraviolet (UV) radiation and defective for nucleotide excision repair in vitro. Interestingly, tfb3(ts) failed to grow on medium containing caffeine. A comprehensive pairwise two-hybrid analysis between yeast TFIIH subunits identified novel interactions between Rad3 and Tfb3, Tfb4 and Ssl1, as well as Ssl2 and Tfb2. These interactions have facilitated a more complete model of the structure of TFIIH and the nucleotide excision repairosome.

Abstract

Three lines of evidence have converged on a multiprotein Mediator complex as a conserved interface between gene-specific regulatory proteins and the general transcription apparatus of eukaryotes. Mediator was discovered as an activity required for transcriptional activation in a reconstituted system from yeast. Upon resolution to homogeneity, the activity proved to reside in a 20-protein complex, which could exist in a free state or in a complex with RNA polymerase II, termed holoenzyme. A second line of evidence came from screens in yeast for mutations affecting transcription. Two-thirds of Mediator subunits are encoded by genes revealed by these screens. Five of the genetically defined subunits, termed Srbs, were characterized as interacting with the C-terminal domain of RNA polymerase II in vivo, and were shown to bind polymerase in vitro. A third line of evidence has come recently from studies in mammalian transcription systems. Mammalian counterparts of yeast Mediator were shown to interact with transcriptional activator proteins and to play an essential role in transcriptional regulation. Mediator evidently integrates and transduces positive and negative regulatory information from enhancers and operators to promoters. It functions directly through RNA polymerase II, modulating its activity in promoter-dependent transcription. Details of the Mediator mechanism remain obscure. Additional outstanding questions include the patterns of promoter-specificity of the various Mediator subunits, the possible cell-type-specificity of Mediator subunit composition, and the full structures of both free Mediator and RNA polymerase II holoenzyme.

Abstract

Some 30 years ago, following the elucidation of transcriptional control in prokaryotes, attention turned to the corresponding problem in eukaryotes: how are so many genes transcribed in a cell-type-specific, developmentally regulated manner? The answer has been found in two modes of regulation, one involving chromatin and the other the chief transcribing enzyme, RNA polymerase II. Although basic features of the prokaryotic mechanism have been preserved, the demands of eukaryotic transcription control are met by a huge increase in complexity and by the addition of new layers to the transcription apparatus. Discovering the components of this apparatus has been a major theme of research over the past three decades; unravelling the mechanisms is a challenge for the future.

Abstract

RSC is an essential 15 protein nucleosome-remodeling complex from S. cerevisiae. We have identified two closely related RSC members, Rsc1 and Rsc2. Biochemical analysis revealed Rsc1 and Rsc2 in distinct complexes, defining two forms of RSC. Genetic analysis has shown that Rsc1 and Rsc2 possess shared and unique functions. Rsc1 and Rsc2 each contain two bromodomains, a bromo-adjacent homology (BAH) domain, and an AT hook. One of the bromodomains, the BAH domain, and the AT hook are each essential for Rsc1 and Rsc2 functions, although they are not required for assembly into RSC complexes. Therefore, these domains are required for RSC function. Additional genetic analysis provides further evidence that RSC function is related to transcriptional control.

Abstract

The structure of an actively transcribing complex, containing yeast RNA polymerase II with associated template DNA and product RNA, was determined by electron crystallography. Nucleic acid, in all likelihood the "transcription bubble" at the active center of the enzyme, occupies a previously noted 25 A channel in the protein structure. Details are indicative of a roughly 90 degrees bend of the DNA between upstream and downstream regions. The DNA apparently lies entirely on one face of the polymerase, rather than passing through a hole to the opposite side, as previously suggested.

Abstract

Appropriate treatment of X-ray diffraction from an unoriented 18-heavy atom cluster derivative of a yeast RNA polymerase II crystal gave significant phase information to 5 A resolution. The validity of the phases was shown by close similarity of a 6 A electron density map to a 16 A molecular envelope of the polymerase from electron crystallography. Comparison of the 6 A X-ray map with results of electron crystallography of a paused transcription elongation complex suggests functional roles for two mobile protein domains: the tip of a flexible arm forms a downstream DNA clamp; and a hinged domain may serve as an RNA clamp, enclosing the transcript from about 8-18 residues upstream of the 3'-end in a tunnel.

Abstract

Nucleosomes have long been known to inhibit DNA transactions on chromosomes and a remarkable abundance of multiprotein complexes that either enhance or relieve this inhibition have been described. Most is known about chromatin-remodeling complexes that perturb nucleosome structure.

Abstract

DNA double-strand breaks are created by ionizing radiation or during V(D)J recombination, the process that generates immunological diversity. Breaks are repaired by an end-joining reaction that requires DNA-PKCS, the catalytic subunit of DNA-dependent protein kinase. DNA-PKCS is a 460 kDa serine-threonine kinase that is activated by direct interaction with DNA. Here we report its structure at 22 A resolution, as determined by electron crystallography. The structure contains an open channel, similar to those seen in other double-stranded DNA-binding proteins, and an enclosed cavity with three openings large enough to accommodate single-stranded DNA, with one opening adjacent to the open channel. Based on these structural features, we performed biochemical experiments to examine the interactions of DNA-PKCS with different DNA molecules. Efficient kinase activation required DNA longer than 12 bp, the minimal length of the open channel. Competition experiments demonstrated that DNA-PKCS binds to double- and single-stranded DNA via separate but interacting sites. Addition of unpaired single strands to a double-stranded DNA fragment stimulated kinase activation. These results suggest that activation of the kinase involves interactions with both double- and single-stranded DNA, as suggested by the structure. A model for how the kinase is regulated by DNA is described.

Abstract

Single particles of the mediator of transcriptional regulation (Mediator) and of RNA polymerase II holoenzyme were revealed by electron microscopy and image processing. Mediator alone appeared compact, but at high pH or in the presence of RNA polymerase II it displayed an extended conformation. Holoenzyme contained Mediator in a fully extended state, partially enveloping the globular polymerase, with points of apparent contact in the vicinity of the polymerase carboxyl-terminal domain and the DNA-binding channel. A similarity in appearance and conformational behavior of yeast and murine complexes indicates a conservation of Mediator structure among eukaryotes.

Abstract

RSC, an abundant, essential chromatin-remodeling complex related to SWI/SNF complex, catalyzes the transfer of a histone octamer from a nucleosome core particle to naked DNA. The newly formed octamer-DNA complex is identical with a nucleosome in all respects. The reaction requires ATP and involves an activated RSC-nucleosome intermediate. The mechanism may entail formation of a duplex displacement loop on the nucleosome, facilitating the entry of exogeneous DNA and the release of the endogenous molecule.

Abstract

Deletion of any one of three subunits of the yeast Mediator of transcriptional regulation, Med2, Pgd1 (Hrs1), and Sin4, abolished activation by Gal4-VP16 in vitro. By contrast, other Mediator functions, stimulation of basal transcription and of TFIIH kinase activity, were unaffected. A different but overlapping Mediator subunit dependence was found for activation by Gcn4. The genetic requirements for activation in vivo were closely coincident with those in vitro. A whole genome expression profile of a Deltamed2 strain showed diminished transcription of a subset of inducible genes but only minor effects on "basal" transcription. These findings make an important connection between transcriptional activation in vitro and in vivo, and identify Mediator as a "global" transcriptional coactivator.

Abstract

Mediator was isolated from yeast on the basis of its requirement for transcriptional activation in a fully defined system. We have now identified three new members of mediator in the low molecular mass range by peptide sequence determination. These are the products of the NUT2, CSE2, and MED11 genes. The product of the NUT1 gene is evidently a component of mediator as well. NUT1 and NUT2 were earlier identified as negative regulators of the HO promoter, whereas mutations in CSE2 affect chromosome segregation. MED11 is a previously uncharacterized gene. The existence of these proteins in the mediator complex was verified by copurification and co-immunoprecipitation with RNA polymerase II holoenzyme.

Abstract

The yeast Saccharomyces cerevisiae contains two related chromatin-remodeling complexes, RSC and SWI/SNF, which are shown to share the actin-related proteins Arp7 and Arp9. Depending on the genetic background tested, arp7 delta and arp9 delta mutants are either inviable or show greatly impaired growth and Swi-/Snf- mutant phenotypes. Unlike swi/snf mutants, viable arp7 delta or arp9 delta mutants have an Spt- phenotype, suggesting that RSC affects transcription. Temperature-sensitive mutations in ARP7 and ARP9 were isolated, and the amino acid changes support the structural relationship of Arp7 and Arp9 to actin. However, site-directed mutations predicted to impair ATP binding or hydrolysis did not detectably affect Arp7 or Arp9 function. Our results suggest that actin-related proteins perform important roles in chromatin-remodeling complexes by virtue of structural rather than enzymatic similarities to actin.

Single-particle selection and alignment with heavy atom cluster-antibody conjugatesPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAJensen, G. J., Kornberg, R. D.1998; 95 (16): 9262-9267

Abstract

A method is proposed for selecting and aligning images of single biological particles to obtain high-resolution structural information by cryoelectron microscopy. The particles will be labeled with multiple heavy atom clusters to permit the precise determination of particle locations and relative orientations even when imaged close to focus with a low electron dose, conditions optimal for recording high-resolution detail. Heavy atom clusters should also allow selection of images free from many kinds of defects, including specimen movement and particle inhomogeneity. Heavy atom clusters may be introduced in a general way by the construction of "adaptor" molecules based on single-chain Fv antibody fragments, consisting of a constant framework region engineered for optimal cluster binding and a variable antigen binding region selected for a specific target. The success of the method depends on the mobility of the heavy atom cluster on the particle, on the precision to which clusters can be located in an image, and on the sufficiency of cluster projections alone to orient and select particles for averaging. The necessary computational algorithms were developed and implemented in simulations that address the feasibility of the method.

Mammalian mediator of transcriptional regulation and its possible role as an end-point of signal transduction pathwaysPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAJiang, Y. W., Veschambre, P., Erdjument-Bromage, H., Tempst, P., Conaway, J. W., Conaway, R. C., Kornberg, R. D.1998; 95 (15): 8538-8543

Abstract

A multiprotein complex isolated from murine cells is identified as a counterpart of the yeast Mediator of transcriptional regulation on the basis of the following: homologs of two subunits of yeast Mediator, Srb7 and Med7, copurify with the complex; peptide sequencing reveals, in addition, homologs of the yeast Mediator subunits Rgr1 and Med6; as with yeast Mediator, the mouse complex binds to the RNA polymerase II C-terminal domain (CTD) and stimulates phosphorylation of the CTD by TFIIH. Peptide sequencing also identifies a component of mouse Mediator as a relative of Ring-3 protein, a mitogen-activated nuclear protein kinase, raising the possibility of Mediator as an end point of signal transduction pathways.

Abstract

X-ray diffraction data from two forms of yeast RNA polymerase II crystals indicate that the two largest subunits of the polymerase, Rpb1 and Rpb2, may have similar folds, as is suggested by secondary structure predictions. DNA may bind between the two subunits with its 2-fold axis aligned to a pseudo 2-fold axis of the protein.

Abstract

RSC, an abundant, essential chromatin-remodeling complex, related to SWI/SNF complex, binds nucleosomes and naked DNA with comparable affinities, as shown by gel shift analysis. The RSC-nucleosome complex is converted in the presence of ATP to a slower migrating form. This activated complex exhibits greatly increased susceptibility to endo- and exonucleases but retains a full complement of histones. Activation persists in the absence of ATP, and on removal of RSC, the nucleosome is released in an altered form, with a diminished electrophoretic mobility, greater sedimentation rate, and marked instability at elevated ionic strength. The reaction is reversible in the presence of RSC and ATP, with conversion of the altered form back to the nucleosome.

Abstract

The three-dimensional structure of wild-type yeast RNA polymerase II has been determined at a nominal resolution of 24 A. A difference map between this structure and that of the polymerase lacking subunits Rpb4 and Rpb7 showed these two subunits forming part of the floor of the DNA-binding (active center) cleft, and revealed a slight inward movement of the protein domain surrounding the cleft. Surface plasmon resonance measurements showed that Rpb4 and Rpb7 stabilize a minimal pre-initiation complex containing promoter DNA, TATA box-binding protein (TBP), transcription factor TFIIB and the polymerase. These findings suggest that Rpb4 and Rpb7 play a role in coupling the entry of DNA into the active center cleft to closure of the cleft. Such a role can explain why these subunits are necessary for promoter-specific transcription in vitro and for a normal stress response in vivo.

Abstract

Two-dimensional (2-D) crystals of yeast RNA polymerase preserved in vitreous ice were studied by electron crystallographic and single-particle techniques. An electron density projection map of the enzyme was calculated from the data, which extended to a resolution of about 12 A, but was unexpectedly weak at resolutions higher than about 20 A. Multivariate statistics analysis revealed a large amount of variability in unit-cell structure in the polymerase crystals, partially related to high mobility of certain polymerase domains. Those same domains were previously identified as being involved in a conformational transition in the enzyme that controls DNA processivity and access to the active center cleft. Electron microscopic studies of other large multiprotein complexes are likely to require similar approaches to those described here.

Abstract

Mediator was resolved from yeast as a multiprotein complex on the basis of its requirement for transcriptional activation in a fully defined system. Three groups of mediator polypeptides could be distinguished: the products of five SRB genes, identified as suppressors of carboxy-terminal domain (CTD)-truncation mutants; products of four genes identified as global repressors; and six members of a new protein family, termed Med, thought to be primarily responsible for transcriptional activation. Notably absent from the purified mediator were Srbs 8, 9, 10, and 11, as well as members of the SWI/SNF complex. The CTD was required for function of mediator in vitro, in keeping with previous indications of involvement of the CTD in transcriptional activation in vivo. Evidence for human homologs of several mediator proteins, including Med7, points to similar mechanisms in higher cells.

Abstract

Minimal templates were devised for the efficient generation of yeast RNA polymerase II transcription elongation complexes. A 33-base pair DNA with a 15-residue dC tail at one 3'-end supported the formation of a complex containing the polymerase paused at nucleotide 11 of the duplex region and an RNA of 14-16 residues. The same template could yield an arrested complex with the enzyme at nucleotide 13-15 and RNA of 15-17 residues. These complexes were stable for at least a week under various conditions and could be resolved by gel electrophoresis or purified by ion exchange chromatography. The purified paused complex formed crystals capable of x-ray diffraction to 3.5 A resolution. The complex remained active in the crystal and, in the presence of nucleoside triphosphates, could efficiently extend the transcript in situ.

Abstract

A new two-dimensional crystal form of yeast RNA polymerase II was obtained in which the conformation of the enzyme appears "open", allowing entry of DNA, as required for the initiation of transcription. By contrast, a previous crystal form contained the enzyme in a "closed" conformation, appropriate for retention of DNA during RNA chain elongation. Interaction with two polymerase subunits, Rpb4 and Rpb7, favors the closed conformation, and binding of general transcription factor TFIIE may do so as well. The effect of Rpb4 and Rpb7, together with previous biochemical evidence, leads to the conclusion that the open to closed transition is a crucial step in the transcription initiation process.

Abstract

Genes for the Tfb2, Tfb3, and Tfb4 subunits of yeast RNA polymerase transcription factor IIH (TFIIH) are described. All three genes are essential for cell viability, and antibodies against Tfb3 specifically inhibit transcription in vitro. A C-terminal deletion of Tfb2 caused a defect in nucleotide excision repair, as shown by UV sensitivity of the mutant strain and loss of nucleotide excision repair activity in cell extracts (restored by the addition of purified TFIIH). An interaction between Tfb3 and the Kin28 subunit of TFIIH was detected by the two-hybrid approach, consistent with a role for Tfb3 in linking kinase and core domains of the factor. The deduced amino acid sequence of Tfb2 is similar to that of the 52-kDa subunit of human TFIIH, while Tfb3 is identified as a RING finger protein homologous to the 36-kDa subunit of murine CAK (cyclin-dependent kinase activating kinase) and to the 32-kDa subunit of human TFIIH. Tfb4 is homologous to p34 of human TFIIH and is identified as the weakly associated 37-kDa subunit of the yeast factor. These and other findings reveal a one-to-one correspondence and high degree of sequence similarity between the entire set of yeast and human TFIIH polypeptides.

Abstract

Protocols are presented for the preparation of a fully defined yeast RNA polymerase II transcription system, consisting of essentially pure TFIIB, -E, -F, and -H, TATA-binding protein, RNA polymerase II, and mediator of transcriptional regulation. This system, comprising 44 polypeptides, is able to initiate transcription at any of a dozen yeast and mammalian promoters thus far tested and responds to a variety of transcriptional activator proteins.

Evidence for a mediator cycle at the initiation of transcriptionPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICASvejstrup, J. Q., Li, Y., Fellows, J., Gnatt, A., Bjorklund, S., Kornberg, R. D.1997; 94 (12): 6075-6078

Abstract

Free and elongating (DNA-bound) forms of RNA polymerase II were separated from yeast. Most cellular polymerase II was found in the elongating fraction, which contained all enzyme phosphorylated on the C-terminal domain and none of the 15-subunit mediator of transcriptional regulation. These and other findings suggest that mediator enters and leaves initiation complexes during every round of transcription, in a process that may be coupled to C-terminal domain phosphorylation.

Abstract

Several eukaryotic multiprotein complexes, including the Saccharomyces cerevisiae Snf/Swi complex, remodel chromatin for transcription. In contrast to the Snf/Swi proteins, Sfh1p, a new Snf5p paralog, is essential for viability. The evolutionarily conserved domain of Sfh1p is sufficient for normal function, and Sfh1p interacts functionally and physically with an essential Snf2p paralog in a novel nucleosome-restructuring complex called RSC (for remodels the structure of chromatin). A temperature-sensitive sfh1 allele arrests cells in the G2/M phase of the cell cycle, and the Sfh1 protein is specifically phosphorylated in the G1 phase. Together, these results demonstrate a link between chromatin remodeling and progression through the cell division cycle, providing genetic clues to possible targets for RSC function.

Abstract

Nucleotide excision repair (NER) is a biochemical process required for the repair of many different types of DNA lesions. In the yeast Saccharomyces cerevisiae, the RAD7, RAD16, and RAD23 genes have been specifically implicated in NER of certain transcriptionally repressed loci and in the nontranscribed strand of transcriptionally active genes. We have used a cell-free system to study the roles of the Rad7, Rad16, and Rad23 proteins in NER. Transcription-independent NER of a plasmid substrate was defective in rad7, rad16, and rad23 mutant extracts. Complementation studies with a previously purified NER protein complex (nucleotide excision repairosome) indicate that Rad23 is a component of the repairosome, whereas Rad7 and Rad16 proteins were not found in this complex. Complementation studies with rad4, rad7, rad16, and rad23 mutant extracts suggest physical interactions among these proteins. This conclusion was confirmed by experiments using the yeast two-hybrid assay, which demonstrated the following pairwise interactions: Rad4 with Rad23, Rad4 with Rad7, and Rad7 with Rad16. Additionally, interaction between the Rad7 and Rad16 proteins was demonstrated in vitro. Our results show that Rad7, Rad16, and Rad23 are required for transcription-independent NER in vitro. This process may involve a unique protein complex which is distinct from the repairosome and which contains at least the Rad4, Rad7, and Rad16 proteins.

Abstract

Yeast Rox3 protein, implicated by genetic evidence in both negative and positive transcriptional regulation, is identified as a mediator subunit by peptide sequence determination and is shown to copurify and co-immunoprecipitate with RNA polymerase II holoenzyme.

Abstract

A novel 15-subunit complex with the capacity to remodel the structure of chromatin, termed RSC, has been isolated from S. cerevisiae on the basis of homology to the SWI/SNF complex. At least three RSC subunits are related to SWI/SNF polypeptides: Sth1p, Rsc6p, and Rsc8p are significantly similar to Swi2/Snf2p, Swp73p, and Swi3p, respectively, and were identified by mass spectrometric and sequence analysis of peptide fragments. Like SWI/SNF, RSC exhibits a DNA-dependent ATPase activity stimulated by both free and nucleosomal DNA and a capacity to perturb nucleosome structure. RSC is, however, at least 10-fold more abundant than SWI/SNF complex and is essential for mitotic growth. Contrary to a report for SWII/SNF complex, no association of RSC (nor of SWI/SNF complex) with RNA polymerase II holoenzyme was detected.

Abstract

A yeast protein has been identified that stimulates basal transcription by RNA polymerase II, binds both single- and double-stranded DNA, and interacts with both a general transcription factor and a transcriptional activator. Phosphorylation appears to regulate these interactions. The gene for the transcriptional stimulatory protein, termed TSP1, was cloned and found to be dispensable for yeast cell viability. The deduced amino acid sequence is similar to that of mammalian coactivator protein PC4.

The yeast GAL11 protein binds to the transcription factor IIE through GAL11 regions essential for its in vivo functionPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICASakurai, H., Kim, Y. J., Ohishi, T., Kornberg, R. D., Fukasawa, T.1996; 93 (18): 9488-9492

Abstract

The GAL11 gene encodes an auxiliary transcription factor required for full expression of many genes in yeast. The GAL11-encoded protein (Gal11p) has recently been shown to copurify with the holoenzyme of RNA polymerase II. Here we report that Gal11p stimulates basal transcription in a reconstituted transcription system composed of recombinant or highly purified transcription factors, TFIIB, TFIIE, TFIIF, TFIIH, and TATA box-binding protein and core RNA polymerase II. We further demonstrate that each of the two domains of Gal11p essential for in vivo function respectively participates in the binding to the small and large subunits of TFIIE. The largest subunit of RNA polymerase II was coprecipitated by anti-hemagglutinin epitope antibody from crude extract of GAL11 wild type yeast expressing hemagglutinintagged small subunit of TFIIE. Such a coprecipitation of the RNA polymerase subunit was seen but in a greatly reduced amount, if extract was prepared from gal11 null yeast. In light of these findings, we suggest that Gal11p stimulates promoter activity by enhancing an association of TFIIE with the preinitiation complex in the cell.

Abstract

Swi/Snf protein was purified previously from the yeast Saccharomyces cerevisiae as an 11-polypeptide complex, including five novel Swp polypeptides. Here we present evidence concerning the role of Swp73p in the function of the complex. Deletion mutants in the SWP73 gene display phenotypes similar to those of swi and snf mutants, and in addition are temperature-sensitive. Swp73p is required for transcriptional activation by full-length glucocorticoid receptor (GR), but not by all GR derivatives. Swp73p is also required for activation with an enhancer element that binds the transcription factors Swi5p and Pho2p, which may underlie the defects in HO expression observed with swi and snf mutants. A single amino acid change in the protein confers phenotypes that are similar to those observed in the swp73 delta strain, but in some cases the two strains behave differently. The extent to which Swp73p is required for assisting transcriptional activation depends on the activator and promoter tested. Homologs of SWP73 are present in S. cerevisiae, Ashbya gossypii, Caenorhabditis elegans, and mice, indicating that SWP73 may belong to a family of related genes encoding proteins with analogous functions.

Abstract

All pairwise interactions of RNA polymerase II and general transcription factors (TF) IIB, E, F, and H have been quantitated by surface plasmon resonance with the use of a Ni2+ chelate on the sensor surface where necessary to attain higher sensitivity. Only 4 of 10 possible interactions were found above the detection limit: TFIIB, -E, and -F binding to RNA polymerase II and TFIIE binding to TFIIH. These four interactions constitute a minimal set for the formation of a transcription initiation complex and may represent the primary interactions involved in assembly of the complex. Point mutations in TFIIB that alter the location of transcription start sites in vivo markedly diminished the affinity of TFIIB binding to RNA polymerase II. Protein blotting revealed an interaction between the largest subunit of TFIIE and third largest subunit of TFIIH, which may be responsible for TFIIE binding to TFIIH.

TFG3/TAF30/ANC1, a component of the yeast SWI/SNF complex that is similar to the leukemogenic proteins ENL and AF-9MOLECULAR AND CELLULAR BIOLOGYCairns, B. R., Henry, N. L., Kornberg, R. D.1996; 16 (7): 3308-3316

Abstract

The SWI1/ADR6, SWI2/SNF2, SWI3, SNF5, and SNF6 gene products are all required for proper transcriptional control of many genes in the yeast Saccharomyces cerevisiae. Genetic studies indicated that these gene products might form a multiprotein SWI/SNF complex important for chromatin transitions preceding transcription from RNA polymerase II promoters. Biochemical studies identified a SWI/SNF complex containing these and at least six additional polypeptides. Here we show that the 29-kDa component of the SWI/SNF complex is identical to TFG3/TAF30/ANC1. Thus, a component of the SWI/SNF complex is also a member of the TFIIF and TFIID transcription complexes. TFG3 interacted with the SNF5 component of the SWI/SNF complex in protein interaction blots. TFG3 is significantly similar to ENL and AF-9, two proteins implicated in human acute leukemia. These results suggest that ENL and AF-9 proteins interact with the SNF5 component of the human SWI/SNF complex and raise the possibility that the SWI/SNF complex is involved in acute leukemia.

Abstract

Transcription factors IIB (TFIIB) and IIE (TFIIE) bound to RNA polymerase II have been revealed by electron crystallography in projection at 15.7 A resolution. The results lead to simple hypotheses for the roles of these factors in the initiation of transcription. TFIIB is suggested to define the distance from TATA box to transcription start site by bringing TATA DNA in contact with polymerase at that distance from the active center of the enzyme. TFIIE is suggested to participate in a key conformational switch occurring at the active center upon polymerase-DNA interaction.

Abstract

The location of the CTD in the structure of RNA polymerase II has been determined by electron crystallography at 16 A resolution. Difference maps between wild-type enzyme and that lacking the CTD, or with an antibody fragment bound in place of the CTD, disclose the site of attachment of the CTD to the polymerase. Appropriate display of the polymerase structure reveals the CTD as an element projecting from this site of attachment into solution. A low relative density and large volume of this element identify the CTD as a conformationally mobile region.

Abstract

Both 45- and 47-kDa subunits of TFIIK, a subcomplex of RNA polymerase II general transcription factor TFIIH, are encoded by the yeast cyclin gene CCL1. In all likelihood, these two subunits individually form cyclin-dependent kinase/cyclin dimers with Kin28 protein, a key enzyme in phosphorylation of the C-terminal domain of RNA polymerase II concomitant with transcription.

Abstract

Sin4 and Rgr1 proteins, previously shown by genetic studies to play both positive and negative roles in the transcriptional regulation of many genes, are identified here as components of mediator and RNA polymerase II holoenzyme complexes. Results with Sin4 deletion and Rgr1 truncation strains indicate the association of these proteins in a subcomplex comprising Sin4, Rgr1, Gal11, and a 50-kDa polypeptide. Taken together with the previous genetic evidence, our findings point to a role of the mediator in repression as well as in transcriptional activation.

Abstract

Three G1 cyclins, CLN1, CLN2, and CLN3, have been identified in the budding yeast Saccharomyces cerevisiae. G1 cyclins are essential, albeit functionally redundant, rate-limiting activators of cell cycle initiation. We have isolated dosage-dependent suppressor genes (designated HMD genes) of the mating defect caused by CLN3-2, a dominant mutation in CLN3, HMD2 and HMD3 are identical to STE4 and STE5, respectively, HMD1 is an essential gene that encodes a protein containing a putative RNA binding domain. Overproduction of HMD1 results in a relatively specific reduction in the level of the CLN3 or CLN3-2 transcript. This reduction occurs subsequent to transcription initiation of CLN3 since overexpression of HMD1 did not affect expression of a heterologous transcript from the CLN3 promoter but did result in a reduction of CLN3 transcript expressed from a heterologous promoter. HMD1 has at least one essential role independent of its effect on CLN3 since HMD1 remains essential for viability in the absence of a functional CLN3 gene.

Abstract

Although the mechanisms of transcriptional regulation by RNA polymerase II are apparently highly conserved from yeast to man, the identification of a yeast TATA-binding protein (TBP)-TBP-associated factor (TAFII) complex comparable to the metazoan TFIID component of the basal transcriptional machinery has remained elusive. Here, we report the isolation of a yeast TBP-TAFII complex which can mediate transcriptional activation by GAL4-VP16 in a highly purified yeast in vitro transcription system. We have cloned and sequenced the genes encoding four of the multiple yeast TAFII proteins comprising the TBP-TAFII multisubunit complex and find that they are similar at the amino acid level to both human and Drosophila TFIID subunits. Using epitope-tagging and immunoprecipitation experiments, we demonstrate that these genes encode bona fide TAF proteins and show that the yeast TBP-TAFII complex is minimally composed of TBP and seven distinct yTAFII proteins ranging in size from M(r) = 150,000 to M(r) = 25,000. In addition, by constructing null alleles of the cloned TAF-encoding genes, we show that normal function of the TAF-encoding genes is essential for yeast cell viability.

Abstract

Research on the interplay between chromatin and transcription has progressed along three lines during the past year. Evidence has been reported for disruption of nucleosomes by transcriptional regulatory proteins in cell-free systems; displacement of the histone octamer during transcription has been conclusively demonstrated; and insights into transcriptional repression by heterochromatin have been gained from studies of silent mating loci and telomeres in yeast.

Abstract

The essential TFB1 and SSL1 genes of the yeast Saccharomyces cerevisiae encode two subunits of the RNA polymerase II transcription factor TFIIH (factor b). Here we show that extracts of temperature-sensitive mutants carrying mutations in both genes (tfb1-101 and ssl1-1) are defective in nucleotide excision repair (NER) and RNA polymerase II transcription but are proficient for base excision repair. RNA polymerase II-dependent transcription at the CYC1 promoter was normal at permissive temperatures but defective in extracts preincubated at a restrictive temperature. In contrast, defective NER was observed at temperatures that are permissive for growth. Additionally, both mutants manifested increased sensitivity to UV radiation at permissive temperatures. The extent of this sensitivity was not increased in a tfb1-101 strain and was only slightly increased in a ssl1-1 strain at temperatures that are semipermissive for growth. Purified factor TFIIH complemented defective NER in both tfb1-101 and ssl1-1 mutant extracts. These results define TFB1 and SSL1 as bona fide NER genes and indicate that, as is the case with the yeast Rad3 and Ss12 (Rad25) proteins, Tfb1 and Ssl1 are required for both RNA polymerase II basal transcription and NER. Our results also suggest that the repair and transcription functions of Tfb1 and Ssl1 are separable.

Abstract

Two-dimensional (2D) crystals of proteins on lipid monolayers can initiate the formation of large three-dimensional (3D) crystals suitable for X-ray diffraction studies. The role of the 2D crystals in this process has not been firmly established. While it is likely that the 2D crystals serve as nuclei for epitaxial crystal growth, other mechanisms, such as non-specific nucleation induced by the high local concentration of the protein at the surface of the lipid layer, cannot be excluded. Using streptavidin as a model system, we have now firmly established that 3D crystal growth from 2D crystals on lipid layers occurs by epitaxy. We show that 2D crystals of streptavidin (space group C222) on biotinated lipid layers nucleate the growth of a 3D crystal form (space group I4I22) that possesses a structural similarity with the 2D crystal, but have no effect on the growth of 3D crystal forms (I222 and P2(1)) that are unrelated to the 2D crystal. At lower pH, a new 3D crystal form (space group P1), unrelated to the previously described 2D crystals, grew from lipid layers. This discovery initially raised concern about the validity of the epitaxial mechanism, but these concerns were alleviated with the subsequent discovery of a structurally related 2D P1 crystal that grew in similar solution conditions. Some parameters affecting epitaxial growth of both the P1 and I4I22 crystals were investigated, revealing several noteworthy features of the epitaxial growth. (1) 2D crystals are very effective nucleating agents; for instance, the P1 2D crystals can direct the growth of P1 3D crystals even under conditions that favour the growth of other crystal forms. (2) The epitaxial 3D crystal grow very rapidly and at amazingly low protein concentrations; P1 3D crystals can be grown from solutions as low as 10 microM streptavidin. (3) There is no obligate requirement for the deposition of pre-formed 2D crystals; lipid layers alone are equally effective at promoting epitaxial crystal growth.

Abstract

Nucleotide excision repair (NER) in eukaryotes is a biochemically complex process involving multiple gene products. The budding yeast Saccharomyces cerevisiae is an informative model for this process. Multiple genes and in some cases gene products that are indispensable for NER have been isolated from this organism. Homologues of many of these yeast genes are structurally and functionally conserved in higher organisms, including humans. The yeast Rad1/Rad10 heterodimeric protein complex is an endonuclease that is believed to participate in damage-specific incision of DNA during NER. This endonuclease is also required for specialized types of recombination. The products of the RAD3, SSL2(RAD25) SSL1 and TFB1 genes have dual roles in NER and in RNA polymerase II-dependent basal transcription.

Abstract

Yeast TFIIH that is active in transcription can be dissociated into three components: a 5-subunit core, the SSL2 gene product, and a complex of 47 kDa, 45 kDa, and 33 kDa polypeptides that possesses protein kinase activity directed towards the C-terminal repeat domain of RNA polymerase II. These three components can reconstitute fully functional TFIIH, and all three are required for transcription in vitro. By contrast, TFIIH that is highly active in nucleotide excision repair (NER) lacks the kinase complex and instead contains the products of all other genes known to be required for NER in yeast: RAD1, RAD2, RAD4, RAD10, and RAD14. This repairosome is not active in reconstituted transcription in vitro and is significantly more active than any of the constituent polypeptides in correcting defective repair in extracts from strains mutated in NER genes.

Abstract

Transfer of two-dimensional (2-D) crystals formed on lipid layers by suspension from a wire loop is described. This method gives better recovery and better preservation of 2-D crystals than attained in the past. The method has been applied to crystals of yeast RNA polymerase II to enable their analysis in the frozen hydrated state.

Abstract

KIN28, a member of the p34cdc2/CDC28 family of protein kinases, is identified as a subunit of yeast RNA polymerase transcription factor IIH (TFIIH) on the basis of sequence determination, immunological reactivity, and copurification. KIN28 is, moreover, one of three subunits of TFIIK, a subassembly of TFIIH with protein kinase activity directed toward the C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II. Itself a phosphoprotein, KIN28 interacts specifically with the two largest subunits of RNA polymerase II. Previous work of others points to two further associations: KIN28 interacts in vivo with the cyclin CCL1, and KIN28 and CCL1 are homologous to human MO15 and cyclin H, which form the cyclin-dependent kinase-activating kinase (CAK). We show that human CAK possesses the CTD kinase activity characteristic of TFIIH.

Abstract

RNA polymerase transcription factor IIF (TFIIF) is required for initiation at most, if not all, polymerase II promoters. We report here the cloning and sequencing of genes for a yeast protein that is the homolog of mammalian TFIIF. This yeast protein, previously designated factor g, contains two subunits, Tfg1 and Tfg2, both of which are required for transcription, essential for yeast cell viability, and whose sequences exhibit significant similarity to those of the mammalian factor. The yeast protein also contains a third subunit, Tfg3, which is less tightly associated and at most stimulatory to transcription, dispensable for cell viability, and has no known counterpart in mammalian TFIIF. Remarkably, the TFG3 gene encodes yeast TAF30, and furthermore, is identical to ANC1, a gene implicated in actin cytoskeletal function in vivo (Welch and Drubin 1994). Tfg3 is also a component of the recently described mediator complex (Kim et al. 1994), whose interaction with the carboxy-terminal repeat domain of RNA polymerase II enables transcriptional activation. Deletion of TFG3 results in diminished transcription in vivo.

ISOLATION OF THE YEAST HISTONE OCTAMERPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICALorch, Y., Kornberg, R. D.1994; 91 (23): 11032-11034

Abstract

Procedures for the extraction and purification of the yeast histone octamer are described. Either mechanical disruption, yielding chromatin fragments, or spheroplast formation with subsequent nuclear isolation was employed. A hexahistidine tag was inserted in the N-terminal region of histone H2B, permitting resolution of the histone octamer from high-salt extracts of nuclei or chromatin to near homogeneity. The histone octamer purified in this way was fully active in reconstitution of nucleosomes.

Abstract

Genes encoding both the 66- and the 43-kDa subunits of yeast RNA polymerase II initiation factor a, designated TFA1 and TFA2, have been isolated. Both genes are essential for cell viability. The bacterially expressed gene products could replace factor a in transcription in vitro, and both recombinant subunits were required for activity. The deduced amino acid sequences of the TFA1 and TFA2 gene products were homologous to those of the large and small subunits of human TFIIE, respectively, identifying factor a as the yeast homolog of TFIIE.

Abstract

The Rad2, Rad3, Rad4, and Ss12 proteins are required for nucleotide excision repair in yeast cells and are homologs of four human proteins which are involved in a group of hereditary repair-defective diseases. We have previously shown that Rad3 protein is one of the five subunits of purified RNA polymerase II basal transcription initiation factor b (TFIIH) and that Ss12 protein physically associates with factor b (W.J. Feaver, J.Q. Svejstrup, L. Bardwell, A.J. Bardwell, S. Buratowski, K.D. Gulyas, T.F. Donahue, E.C. Friedberg, and R.D. Kornberg, Cell 75:1379-1387, 1993). Here we show that the Rad2 and Rad4 proteins interact with purified factor b in vitro. Rad2 (a single-stranded DNA endonuclease) specifically interacts with the Tfb1 subunit of factor b, and we have mapped a limited region of the Rad2 polypeptide which is sufficient for this interaction. Rad2 also interacts directly with Ss12 protein (a putative DNA helicase). The binding of Rad2 and Rad4 proteins to factor b may define intermediates in the assembly of the nucleotide excision repair repairosome. Furthermore, the loading of factor b (or such intermediates) onto promoters during transcription initiation provides a mechanism for the preferential targeting of repair proteins to actively transcribing genes.

Abstract

A mediator was isolated from yeast that enabled a response to the activator proteins GAL4-VP16 and GCN4 in a transcription system reconstituted with essentially homogeneous basal factors and RNA polymerase II. The mediator comprised some 20 polypeptides, including the three subunits of TFIIF and other polypeptides cross-reactive with antisera against GAL11, SUG1, SRB2, SRB4, SRB5, and SRB6 proteins. Mediator not only enabled activated transcription but also conferred 8-fold greater activity in basal transcription and 12-fold greater efficiency of phosphorylation of RNA polymerase II by the TFIIH-associated C-terminal repeat domain (CTD) kinase, indicative of mediator-CTD interaction. A holoenzyme form of RNA polymerase II was independently isolated that supported a response to activator proteins with purified basal factors. The holoenzyme proved to consist of mediator associated with core 12-subunit RNA polymerase II.

Abstract

Transcription initiation by RNA polymerase II is effected by an ordered series of general factor interactions with core promoter elements (leading to basal activity) and further regulated by gene-specific factors acting from distal elements. Both the general factor TFIID (refs 2,3), including the constituent TBP (TATA-binding polypeptide) and associated factors, and the interacting factor TFIIB (refs 9-11) have been implicated as targets for various activators. Towards an understanding of the basis for activator function, including the multiplicity of TBP interactions, we have now identified mutations in yeast TBP that selectively block activator (GAL4-VP16)-dependent but not basal transcription. We further show an effect of GAL4-VP16 on TFIIB recruitment to early preinitiation complexes, and that recruitment is disrupted by TBP mutations that impair its interactions with VP16 (L114K), TFIIB (L189K) or an unidentified component (K211L). Thus, GAL4-VP16 function seems to involve both direct interactions with TBP and a corresponding induction (or stabilization) of an activation-specific TBP-TFIIB-promoter complex.

YEAST RADS PROTEIN BINDS DIRECTLY TO BOTH SSL2 AND SSL1 PROTEINS - IMPLICATIONS FOR THE STRUCTURE AND FUNCTION OF TRANSCRIPTION/REPAIR FACTOR-BPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICABardwell, L., Bardwell, A. J., Feaver, W. J., Svejstrup, J. Q., Kornberg, R. D., Friedberg, E. C.1994; 91 (9): 3926-3930

Abstract

The RAD3 and SSL2 gene products are essential proteins that are also required for the nucleotide excision repair pathway. We have recently demonstrated that the RAD3 gene product along with the SSL1 and TFB1 gene products are subunits of RNA polymerase II basal transcription factor b. Additionally, the SSL2 gene product physically interacts with purified factor b. Here we combine an in vitro immunoprecipitation assay and an in vivo genetic assay to demonstrate a series of pairwise protein-protein interactions involving these components. RAD3 protein binds directly to both SSL2 protein and SSL1 protein in vitro. SSL1 protein interacts with itself and with RAD3 and TFB1 proteins in living yeast cells. An N-terminal, possibly noncatalytic, domain of SSL2 protein is sufficient for the factor b-SSL2 interaction, and a product of a DNA repair-defective allele of SSL2 is not defective in binding to factor b. We present genetic evidence suggesting that the DNA-repair function of SSL2 protein is not dependent on its essential function.

INTERPLAY OF POSITIVE AND NEGATIVE EFFECTORS IN FUNCTION OF THE C-TERMINAL REPEAT DOMAIN OF RNA-POLYMERASE-IIPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICALi, Y., Kornberg, R. D.1994; 91 (6): 2362-2366

Abstract

RNA polymerase II lacking a C-terminal domain (CTD) was active in transcription with purified proteins from yeast but failed to support transcription in a yeast extract. CTD dependence could be reconstituted in the purified system by addition of two fractions from the extract. An inhibitory fraction abolished transcription by both wild-type and CTD-less RNA polymerases; a stimulatory fraction restored activity of the wild-type polymerase but had a much lesser effect on the CTD-less enzyme. Parallel results were obtained with the use of a kinase inhibitor that prevents phosphorylation of the CTD by RNA polymerase II initiation factor b. The kinase inhibitor abolished transcription by wild-type polymerase in yeast extract but had no significant effect in the purified system. The requirement for both the CTD and kinase action for transcription in an extract indicates that CTD phosphorylation is involved in opposing the negative effector in the extract. Factor b must play a role(s) in addition to phosphorylation of the CTD because it was still required for transcription with polymerase lacking a CTD in the purified system.

Abstract

Nucleotide-excision repair (NER) is an important cellular defence mechanism against mutagenesis and carcinogenesis. The essential yeast genes RAD3 (ref. 2) and SSL2 (RAD25), homologues of the human xeroderma pigmentosum genes XPD and XPB respectively, have been implicated in NER in yeast. The products of these genes are also subunits of (Rad3 protein) or associate with (Ssl2 protein) purified yeast RNA polymerase II transcription initiation factor b, the counterpart of human TFIIH. Rad3 and Ssl2 proteins may participate directly in NER. Alternatively, they may function exclusively as transcription factors that support NER by influencing the expression of other NER genes. Here we show that defective NER in rad3 mutant extracts can be specifically complemented by purified transcription factor b. Similarly, defective NER in ssl2 mutant extracts is corrected by purified factor b/Ssl2 complex. These results support a direct role of factor b during NER in yeast. Hence, factor b (TFIIH) has a dual role in transcription and NER.

Abstract

A complex containing the products of the SWI1/ADR6, SWI2/SNF2, SWI3, SNF5, and SNF6 genes and four additional polypeptides has been purified from extracts of the yeast Saccharomyces cerevisiae. Physical association of these proteins was demonstrated by copurification and coimmunoprecipitation. A potent DNA-dependent ATPase copurified with the complex, and this activity was evidently associated with SWI2/SNF2.

Abstract

The growth of three-dimensional protein crystals is seeded by two-dimensional crystals formed on lipid layers. Such crystallization occurs faster and at lower precipitant and protein concentrations than conventional crystal growth. This approach may also allow the crystallization of proteins that resist attempts at crystal growth by other means.

Abstract

An RNA polymerase II transcription system was resolved and reconstituted from extracts of Schizosaccharomyces pombe. Exchange with components of a Saccharomyces cerevisiae system was undertaken to reveal the factor or factors responsible for the difference in location of the transcription start site, about 30 base pairs and 40 to 120 base pairs downstream of the TATA box in S. pombe and S. cerevisiae, respectively. Two components, counterparts of human transcription factor IIF (TFIIF) and TFIIH, could be exchanged individually between systems without effect on the start site. Three components, counterparts of human TFIIB, TFIIE, and RNA polymerase II, could not be exchanged individually but could be swapped in the pairs TFIIE-TFIIH and TFIIB-RNA polymerase II, which demonstrates that there are functional interactions between these components. Moreover, exchange of the latter pair shifted the starting position, which shows that TFIIB and RNA polymerase II are solely responsible for determining the start site of transcription.

Abstract

Yeast RNA polymerase II initiation factor b, homolog of human TFIIH, is a protein kinase capable of phosphorylating the C-terminal repeat domain of the polymerase; it possesses a DNA-dependent ATPase activity as well. The 85 kd and 50 kd subunits of factor b are now identified as RAD3 and SSL1 proteins, respectively; both are known to be involved in DNA repair. Factor b interacts specifically with another DNA repair protein, SSL2. The ATPase activity of factor b may be due entirely to that associated with a helicase function of RAD3. Factor b transcriptional activity was unaffected, however, by amino acid substitution at a conserved residue in the RAD3 nucleotide-binding domain, suggesting that the ATPase/helicase function is not required for transcription. These results identify factor b as a core repairosome, which may be responsible for the preferential repair of actively transcribed genes in eukaryotes.

CRYSTAL-STRUCTURE OF YEAST TATA-BINDING PROTEIN AND MODEL FOR INTERACTION WITH DNAPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAChasman, D. I., Flaherty, K. M., Sharp, P. A., Kornberg, R. D.1993; 90 (17): 8174-8178

Abstract

The C-terminal 179-aa region of yeast (Saccharomyces cerevisiae) TATA-binding protein (TBP), phylogenetically conserved and sufficient for many functions, formed crystals diffracting to 1.7-A resolution. The structure of the protein, determined by molecular replacement with coordinates from Arabidopsis TBP and refined to 2.6 A, differed from that in Arabidopsis slightly by an angle of about 12 degrees between two structurally nearly identical subdomains, indicative of a degree of conformational flexibility. A model for TBP-DNA interaction is proposed with the following important features: the long dimension of the protein follows the trajectory of the minor groove; two rows of basic residues conserved between the subdomains lie along the edges of the protein in proximity to the DNA phosphates; a band of hydrophobic residues runs down the middle of the groove; and amino acid residues whose mutation alters specificity for the second base of the TATA sequence are juxtaposed to that base.

A POSSIBLE ROLE FOR THE YEAST TATA-ELEMENT-BINDING PROTEIN IN DNA-REPLICATIONPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICALue, N. F., Kornberg, R. D.1993; 90 (17): 8018-8022

Abstract

The TATA-element-binding protein (TBP) is involved in the initiation of transcription by all three eukaryotic RNA polymerases. The following observations implicate TBP in the initiation of DNA replication at yeast chromosomal origins as well: (i) Recombinant yeast TBP binds specifically to functionally important regions of many yeast replication origins in vitro. (ii) TBP-binding sites from RNA polymerase II promoters can activate defective replication origins in vivo. (iii) Point mutations in TBP-binding sites that diminish their affinity for TBP in vitro reduce their ability to support replication in vivo.

Abstract

Binding of yeast transcription factor IID (TFIID) to the adenoviral major late promoter in circular DNA molecules caused a linking number change of less than 0.1. TFIID on its own therefore fails to unwind DNA appreciably, or else it causes both unwinding and compensatory writhing. Highly purified, recombinant yeast TFIID relaxed supercoiled DNA, because of a contaminant of bacterial topoisomerase I. Relaxing activity of topoisomerase I was enhanced by the adenoviral major late promoter, suggesting an instability of the TATA sequence or a destabilizing effect on flanking DNA.

Abstract

Fractionation of yeast whole-cell extract has led to the identification and purification of four novel proteins required for promoter-dependent transcription by RNA polymerase II in vitro. These initiation factors have been shown to be related in structure and function to RNA polymerase II initiation factors purified from mammalian cells. When combined with the well known TATA-binding protein, the entire set of initiation factors proves necessary and sufficient for accurate initiation from a variety of eukaryotic promoters. Studies with this defined transcription system have revealed the existence of additional proteins required for transcriptional activation or derepression by enhancer-binding regulatory proteins.

Abstract

Templates were prepared with either the TATA box or transcription start sites of the yeast CYC1 promoter in a nucleosome. In both cases, initiation in an unfractionated yeast RNA polymerase II transcription system was abolished by the nucleosome. The inhibition appeared to be relieved by the activator protein Gal4-VP16 binding to a site upstream of the promoter. Inhibition was not relieved, however, in a transcription system reconstituted from purified components, indicating a requirement for additional factors for the effect of Gal4-VP16.

Abstract

Yeast RNA polymerase II initiation factor e was purified to homogeneity and identified by biochemical criteria as the counterpart of human transcription factor IIB. Factor e was essential for initiation of transcription from yeast and mammalian promoters in a reconstituted yeast transcription system. Activity resided in a single polypeptide of approximately 41 kDa, identified by peptide sequence analysis as the product of the SUA7 gene. Factor e interacted specifically with RNA polymerase II, consistent with a proposed role in determining the start site of transcription.

Abstract

RNA polymerase II initiation factor a was purified to apparent homogeneity from yeast whole cell extract and consisted of two highly charged polypeptides with apparent masses of 66 and 43 kDa. Separation and renaturation of the subunits showed that both were required for transcription activity. The native mass of factor a was estimated to be 240-260 kDa by gel filtration, but its sedimentation rate in a glycerol gradient was similar to that of a much smaller globular protein, suggesting an extended conformation. Factor a was required for utilization of six different eukaryotic promoters in vitro, indicating a general role in promoter-directed transcription by yeast RNA polymerase II.

Abstract

Yeast RNA polymerase II general initiation factor g was purified to near homogeneity on the basis of its function in a reconstituted transcription system. Polypeptides of 30, 54, and 105 kDa co-purified with transcriptional activity, forming a complex with a mass of 300 kDa as judged by gel filtration, but only 100 kDa based on sedimentation in glycerol gradients, suggesting an elongated shape. Transcription activity could be reconstituted after separation of the three polypeptides under denaturing conditions; the 54- and 105-kDa subunits were both essential, while the 30-kDa subunit was slightly stimulatory. Factor g was required for initiation at all promoters tested, including those from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and adenovirus. Factor g can stably associate with RNA polymerase II, as shown by cosedimentation in a glycerol gradient.

Abstract

Yeast RNA polymerase II initiation factors a, b, e, and g were isolated from whole cell extract and found to be sufficient, when combined with bacterially expressed yeast transcription factor (TF) IID, to enable RNA polymerase II to utilize nine different eukaryotic promoters in vitro, and to initiate transcription at sites used in vivo. The purified factors did not contain the previously described transcription factor IIA (TFIIA). TFIIA failed to substitute for any purified factor or to stimulate transcription with the complete set of factors, indicating that its function in crude extracts is primarily as an anti-inhibitor.

Abstract

Yeast RNA polymerase II initiation factor b copurifies with three polypeptides of 85, 73, and 50 kilodaltons and with a protein kinase that phosphorylates the carboxyl-terminal repeat domain (CTD) of the largest polymerase subunit. The gene that encodes the 73-kilodalton polypeptide, designated TFB1, was cloned and found to be essential for cell growth. The deduced protein sequence exhibits no similarity to those of protein kinases. However, the sequence is similar to that of the 62-kilodalton subunit of the HeLa transcription factor BFT2, suggesting that this factor is the human counterpart of yeast factor b. Immunoprecipitation experiments using antibodies to the TFB1 gene product demonstrate that the transcriptional and CTD kinase activities of factor b are closely associated with an oligomer of the three polypeptides. Photoaffinity labeling with 3'-O-(4-benzoyl)benzoyl-ATP (adenosine triphosphate) identified an ATP-binding site in the 85-kilodalton polypeptide, suggesting that the 85-kilodalton subunit contains the catalytic domain of the kinase.

SIMPLE DERIVATION OF TFIID-DEPENDENT RNA POLYMERASE-II TRANSCRIPTION SYSTEMS FROM SCHIZOSACCHAROMYCES POMBE AND OTHER ORGANISMS, AND FACTORS REQUIRED FOR TRANSCRIPTIONAL ACTIVATIONPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAFlanagan, P. M., Kelleher, R. J., Tschochner, H., Sayre, M. H., Kornberg, R. D.1992; 89 (16): 7659-7663

Abstract

Resolution of whole cell extract through two chromatographic steps yields a single protein fraction requiring only the addition of TFIID for the initiation of transcription at RNA polymerase II promoters. This approach allows the convenient generation of RNA polymerase II transcription systems from Saccharomyces cerevisiae, human lymphocytes, and Schizosaccharomyces pombe. TFIIDs from all three organisms are interchangeable among all three systems. The S. cerevisiae and Sch. pombe systems support effects of acidic activator proteins, provided a further protein fraction from S. cerevisiae is supplied. This further fraction is distinct from the mediator of transcriptional activation described previously and represents a second component in addition to general initiation factors that may facilitate a response to acidic activators.

ORDER OF ACTION OF COMPONENTS IN THE YEAST PHEROMONE RESPONSE PATHWAY REVEALED WITH A DOMINANT ALLELE OF THE STE11-KINASE AND THE MULTIPLE PHOSPHORYLATION OF THE STE7-KINASEGENES & DEVELOPMENTCairns, B. R., Ramer, S. W., Kornberg, R. D.1992; 6 (7): 1305-1318

Abstract

The signal transduction pathway that mediates the response of haploid yeast cells to peptide mating pheromones involves several components including the protein kinases STE7 and STE11. We have isolated and characterized a dominant allele of the STE11 gene and have demonstrated that expression of an amino-terminally truncated form of STE11 protein causes constitutive activation of the mating pathway. Expression of this dominant STE11 allele also restored mating ability to certain sterile strains. In conjunction with the results of others, our epistasis results establish the following order of action of pathway components: STE2, GPA1(SCG1), STE4, STE5, STE11, STE7, STE12. Transduction of the signal from STE11 to STE7 may involve phosphorylation because STE7 displays several phosphorylation forms, and STE7 is multiply phosphorylated in response to either pheromone or coexpression of dominant STE11 protein. Further signal propagation appears to require STE7 protein kinase activity, because a catalytically impaired STE7 mutant is defective in the mating response.

Abstract

Previous work showed that human TFIID fails to support yeast cell growth, although it is nearly identical to yeast TFIID in a carboxy-terminal region of the molecule that suffices for basal, TATA-element-dependent transcription in vitro. These and other findings raised the possibility that TFIID participates in species-specific interactions, possibly with mediator factors, required for activated transcription. Here, we report that human TFIID and amino-terminally truncated derivatives of yeast TFIID are fully functional in support of both basal transcription and the response to acidic activator proteins in a yeast in vitro transcription system. Conversely, and in contrast to previously published results, yeast TFIID supports both basal and activated transcription in reactions reconstituted with human components. This functional interchangeability of yeast and human TFIIDs argues strongly against species specificity with regard to TFIID function in basal transcription and the response to acidic activator proteins. In addition, our results suggest that any intermediary factors between acidic activators and TFIID are conserved from yeast to man.

Abstract

A kinase activity specific for the C-terminal repeat domain (CTD) of RNA polymerase II is associated with nearly homogeneous yeast general initiation factor b by three criteria: cofractionation on the basis of size and charge and coinactivation by mild heat treatment. The kinase phosphorylates the CTD at multiple sites in a processive manner. Factor b may possess a DNA-dependent ATPase activity as well. Both kinase and DNA-dependent ATPase activities exhibit the same nucleotide requirements as previously demonstrated for the initiation of transcription. These results support the idea that phosphorylation of the CTD lies on the pathway of transcription initiation and identify a catalytic activity of a general factor essential for the initiation process.

Abstract

Heat treatment of yeast nuclear extracts abolished the capacity to initiate transcription at RNA polymerase II promoters. Activity was restored by the addition of both recombinant yeast TFIID and partially purified factor b, a yeast fraction shown previously to be required for polymerase II transcription. On the basis of this assay with heat-treated extract, factor b was purified to virtual homogeneity. The factor appears to comprise polypeptides of approximately 85, 75, and 50 kDa, since these three polypeptides co-purify with activity, and since a native mass of about 200 kDa is estimated from glycerol gradient sedimentation and gel filtration.

Abstract

A mutant form of yeast RNA polymerase II that lacks the fourth and seventh largest subunits, referred to as pol II delta 4/7, crystallized on positively charged lipid layers. Both single-layered (two-dimensional) crystals and several multi-layered crystal forms were obtained. The two-dimensional crystals, preserved in negative stain, diffracted strongly to about 1/20 A-1 and more weakly to 1/13 A-1 resolution. A projection map computed from averaged Fourier transforms revealed four pol II delta 4/7 complexes per unit cell and further revealed a cleft on the surface of the complex similar to that previously observed in the structure of Escherichia coli RNA polymerase. One of the multi-layered crystal forms, preserved in negative stain, diffracted strongly beyond 1/15 A-1 resolution. Coherent diffraction from the multi-layered crystal is indicative of protein-protein interactions between layers and ordering in the third dimension.

Abstract

The structure of yeast RNA polymerase II has been determined by three-dimensional reconstruction from electron micrographs of two-dimensional crystals at approximately 16 A resolution. The most prominent feature of the structure is an arm of protein density surrounding a channel about 25 A in diameter, similar to that found previously for E. coli RNA polymerase. The 25 A-diameter channel bifurcates on one face of the protein, connecting with a 25 A-wide groove and with a channel about half as wide. The 25 A channel and groove, and the narrow channel, may bind double- and single-stranded nucleic acids, respectively. A finger of protein density projecting from the molecule adjacent to the arm-like feature may represent the C-terminal domain of the largest subunit. These results provide a structural basis for analyses of the transcription process and its regulation.

Abstract

Electron crystallographic analysis of two-dimensional crystals grown on lipid layers at the air/water interface has been limited by loss or damage during transfer of the crystals to an electron microscope support grid. Two methods of transfer are described which are applicable on a small scale (10 microliters of protein solution) and which give greatly improved results for streptavidin crystals on biotinylated lipid layers. In the first method, a hydrophobic grid surface was produced by coating a carbon support film with a thin layer of SiO2, followed by alkylation with dimethyloctadecylchlorosilane. The transfer efficiency of protein crystals approached 50% coverage of the alkylated grid surface. The degree of order of crystals transferred to the alkylated grid surface and preserved in negative stain was significantly improved over that of crystals transferred directly to a carbon support film. In the second method, crystals at the air/water interface were transferred to a holey carbon support film. The efficiency of transfer across the holes was virtually 100% as nearly every hole was completely covered with crystals. After preservation of the crystals in 1% glucose and cooling to liquid nitrogen temperature, electron diffraction was obtained that extended to 1/2.8 A-1 resolution. This demonstrates that two-dimensional crystals grown on lipid layers at the air/water interface can be sufficiently well-ordered, even after transfer to a support grid, to yield high-resolution structural information.

Abstract

Activator proteins bind to enhancer DNA elements and stimulate the initiation of transcription. It has been proposed that activators contact general initiation factors at a promoter, and evidence for such direct interaction has been obtained. Studies of transcription in vitro, however, have suggested that activators might function through an intermediary molecule(s) distinct from the general factors. In the first of these studies, we exploited the finding that one activator could inhibit transcription stimulated by a second activator (activator interference or 'squelching'). This inhibition, which is attributed to competition between the activators for a common target factor, could not be relieved by addition of a large excess of general initiation factors, suggesting that the target for which activators compete is distinct from these factors. Similar conclusions came from the observation that TFIID's expressed from cloned genes fail to replace partially purified 'natural' TFIID fractions in supporting activation, evidently because they lacked some component present in the impure fractions. While these lines of evidence for a novel 'mediator' of activation were negative, we also showed that a partially purified fraction from yeast would reverse activator interference. This positive effect of a presumptive mediator provided an assay for its activity, but its role in activation was still only inferred. We now present direct evidence for a mediator which is required for stimulation of transcription in vitro by the activators GAL4-VP16 and GCN4, but which has no effect on transcription in the absence of activator protein.

Abstract

Streptavidin forms two-dimensional crystals when specifically bound to layers of biotinylated lipids at the air/water interface. The three-dimensional structure of streptavidin determined from the crystals by electron crystallography corresponds well with the structure determined by x-ray crystallography. Comparison of the electron and x-ray crystallographic structures reveals the occurrence of free biotin-binding sites on the surface of the two-dimensional crystals facing the aqueous solution. The free biotin-binding sites could be specifically labeled with biotinylated ferritin. The streptavidin/biotinylated lipid system may provide a general approach for the formation of two-dimensional crystals of biotinylated macromolecules.

Abstract

RNA polymerase II lacking the fourth and seventh largest subunits (pol II delta 4/7) was purified from Saccharomyces cerevisiae strain rpb-4, in which the gene for the fourth largest subunit is deleted. pol II delta 4/7 was indistinguishable from wild-type pol II (holoenzyme) in promoter-independent initiation/chain elongation activity (400-800 nmol of nucleotide incorporated/10 min/mg of protein at 22 degrees C), in rate of chain elongation (20-25 nucleotides/s), and in the recognition of pause sites in the DNA template. In contrast to pol II holoenzyme, pol II delta 4/7 was inactive in promoter-directed initiation of transcription in vitro. The addition of an equimolar complex of the fourth and seventh largest subunits, purified from pol II holoenzyme by ion-exchange chromatography in the presence of urea, restored promoter-directed initiation activity to pol II delta 4/7. The transcriptional activator protein Gal4-VP16 could also elicit promoter-directed initiation by pol II delta 4/7 from a promoter with a Gal4 binding site. Complementation was observed between extracts of strain rpb-4, lacking the fourth largest subunit, and strain Y260-1, with a defect in the largest subunit. These extracts were individually inactive, but a mixture would support promoter-directed initiation. The fourth and seventh largest subunits may, therefore, shuttle between polymerase molecules.

Abstract

Two-dimensional crystals of yeast RNA polymerase A (I) were obtained by interaction with positively charged lipid layers. The analysis of single molecular images of lipid-bound RNA polymerases showed that the enzyme was preferentially oriented by the lipid phase, which probably facilitated crystallization. Electron micrographs of the crystals revealed a rectangular unit cell 25.8 nm by 45.6 nm in size containing four RNA polymerase dimers related by P22(1)2(1) symmetry. The projection map showed, at about 2.5 nm resolution, two different views of the enzyme characterized by two bent arms, which appeared to cross at one end. These arms are likely to contain the A190 and A135 subunits and delimit a 3 to 4 nm wide groove. Additional structural features were observed and compared to the Escherichia coli enzyme.

INTERCHANGEABLE RNA-POLYMERASE-I AND POLYMERASE-II ENHANCERSPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICALorch, Y., Lue, N. F., Kornberg, R. D.1990; 87 (21): 8202-8206

Abstract

The RNA polymerase I (pol I) enhancer of Saccharomyces cerevisiae contains at least three elements commonly associated with RNA polymerase II (pol II) enhancers, binding sites for the transcriptional activators general regulatory factor 2 and autonomously replicating sequence-binding factor I, and a thymidine-rich element. When the particular form of the thymidine-rich element found in the pol I enhancer was placed in front of a pol II promoter, transcription was stimulated 43-fold, comparable to the effect of a powerful pol II activator such as Gal4. Conversely, when two copies of a thymidine-rich element from a pol II enhancer were placed upstream of a pol I promoter, transcription was stimulated 38-fold. This functional reciprocity of pol I and II enhancers may reflect similarities in the mechanisms of transcriptional activation. The pol I enhancer also contains an element that appears to be pol I-specific and prevent the activation of pol II.

Abstract

Nuclear extracts from Saccharomyces cerevisiae support initiation by RNA polymerase I at the 35 S rRNA promoter. Initiation occurs at the same site and is stimulated by the polymerase I enhancer element to about the same extent in vitro as in vivo.

Abstract

Fractionation of a yeast nuclear extract reveals at least four factors required in addition to RNA polymerase II for accurate initiation of transcription. One of these factors can be replaced by HeLa transcription factor IID or by its yeast counterpart expressed in Escherichia coli. Each of the remaining three factors can be replaced by a fraction from yeast whole cell extract, facilitating further purification of the factors.

Abstract

One gene activator protein may interfere with the effects of another in eukaryotic cells. We report here that a hybrid yeast-herpes gene activator protein inhibits transcriptional activation by a thymidine-rich DNA element in yeast. This example of activator interference can be faithfully reproduced in vitro. Interference is reversed by a partially purified yeast component, but not by RNA polymerase II or various polymerase II transcription factors. We conclude that the partially purified yeast component is a novel factor, and we suggest this factor mediates the transcriptional activation process.

Abstract

Expression of the yeast Saccharomyces cerevisiae GAL4 protein under its own (galactose-inducible) control gave 5 to 10 times the level of protein observed when the GAL4 gene was on a high-copy plasmid. Purification of GAL4 by a procedure including affinity chromatography on a GAL4-binding DNA column yielded not only GAL4 but also a second protein, shown to be GAL80 by its reaction with an antipeptide antibody. Sequence comparisons of GAL4 and other members of a family of proteins sharing homologous cysteine finger motifs identified an additional region of homology in the middle of these proteins shown by genetic analysis to be important for GAL4 function. GAL4 could be cleaved proteolytically at the boundary of the conserved region, defining internal and carboxy-terminal folded domains.

Abstract

GRF2, an abundant yeast protein of Mr approximately 127,000, binds to the GAL upstream activating sequence (UASG) and creates a nucleosome-free region of approximately 230 bp. Purified GRF2 binds to sequences found in many other UASs, in the 35S rRNA enhancer, at centromeres, and at telomeres. Although GRF2 stimulates transcription only slightly on its own, it combines with a neighboring weak activator to give as much as a 170-fold enhancement. This effect of GRF2 is strongly distance-dependent, declining by 85% when 22 bp is interposed between the GRF2 and neighboring activator sites.

Abstract

Yeast RNA polymerase II was purified to homogeneity by a rapid procedure involving immunoaffinity chromatography. The purified enzyme contained 10 subunits, as reported for conventional preparations, but with no detectable proteolysis of the largest subunit. In assays of initiation of transcription at the yeast CYC1 promoter, the enzyme complemented the deficiency of an extract from a strain that produces a temperature-sensitive polymerase II. Mammalian RNA polymerase II was inactive in this initiation assay. The purified yeast enzyme formed two-dimensional crystals on positively charged lipid layers, as previously found for Escherichia coli RNA polymerase holoenzyme. Image analysis of electron micrographs of crystals in negative stain, which diffracted to about 30-A resolution, showed protein densities of dimensions consistent with those of single polymerase molecules.

Abstract

ABFI (ARS-binding protein I) is a yeast protein that binds specific DNA sequences associated with several autonomously replicating sequences (ARSs). ABFI also binds sequences located in promoter regions of some yeast genes, including DED1, an essential gene of unknown function that is transcribed constitutively at a high level. ABFI was purified by specific binding to the DED1 upstream activating sequence (UAS) and was found to recognize related sequences at several other promoters, at an ARS (ARS1), and at a transcriptional silencer (HMR E). All ABFI-binding sites, regardless of origin, provided weak UAS function in vivo when examined in test plasmids. UAS function was abolished by point mutations that reduced ABFI binding in vitro. Analysis of the DED1 promoter showed that two ABFI-binding sites combine synergistically with an adjacent T-rich sequence to form a strong constitutive activator. The DED1 T-rich element acted synergistically with all other ABFI-binding sites and with binding sites for other multifunctional yeast activators. An examination of the properties of sequences surrounding ARS1 left open the possibility that ABFI enhances the initiation of DNA replication at ARS1 by transcriptional activation.

Abstract

Transcription of the yeast CYC1 promoter fused to a sequence lacking guanosine residues provided a rapid, sensitive assay of initiation by RNA polymerase II in yeast extracts. Initiation was enhanced by yeast and mammalian activator proteins. The adenoviral major late promoter fused to the G-minus sequence was transcribed in yeast extracts with an efficiency comparable to that observed in HeLa extracts, showing that promoters as well as transcription factors are functionally interchangeable across species. Initiation occurred at different sites, approximately 30 and 63 to 69 base pairs downstream of the TATA element of the adenoviral promoter in HeLa and yeast extracts, respectively, distances characteristic of initiation in the two systems in vivo. A component of the transcription system and not the promoter sequence determines the distance to the initiation site.

Abstract

Fusion proteins known to activate transcription in vivo were tested for the ability to stimulate transcription in vitro in a recently developed Saccharomyces cerevisiae RNA polymerase II transcription system. One fusion protein, whose activation domain was derived from the herpesvirus transcriptional activator VP16, gave more than 100-fold stimulation in the in vitro system. The order of effects of the various proteins was the same for transcription in vitro and in vivo, suggesting that the natural mechanism of activation is preserved in vitro.

Abstract

During transcription in E. coli, the DNA-dependent RNA polymerase locates specific promoter sequences in the DNA template, melts a small region containing the transcription start site, initiates RNA synthesis, processively elongates the transcript, and finally terminates and releases the RNA product. Each step is regulated by interactions between the polymerase, the DNA, the nascent RNA, and a variety of regulatory proteins and ligands. The E. coli enzyme contains a catalytic core of two alpha-subunits, one beta- and one beta'-subunit, with relative molecular masses (Mr) of 36,512, 150,619 and 155,162, respectively. The holoenzyme has an additional regulatory subunit, normally sigma, of Mr 70,236. Preparations may also contain the omega-subunit (Mr approximately 10,000), which can be removed without affecting any known properties of the enzyme. Because the amino-acid sequences of the beta- and beta'-subunits are homologous to those of the largest subunits of the yeast, Drosophila and murine RNA polymerases, it seems likely that essential features of the three-dimensional structure and catalytic mechanism of RNA polymerase are also conserved across species. Crystals of RNA polymerase suitable for X-ray analysis have not yet been obtained, but two-dimensional crystals of E. coli RNA polymerase holoenzyme can be grown on positively charged lipid layers. Electron microscopy of these crystals in negative stain shows the enzyme in projection as an irregularly shaped complex approximately 100 x 100 x 160 A in size. We have now determined the three-dimensional structure by electron microscopy of negatively stained, two-dimensional crystals tilted at various angles to the incident electron beam. We find a structure in RNA polymerase similar to the active-site cleft of DNA polymerase I. In the light of functional similarities between these two enzymes, together with other evidence, this probably identifies the active-site region of RNA polymerase.

Abstract

Conversion of the positioned nucleosome array characteristic of the repressed GAL1-GAL10 promoter region to the more accessible conformation of the induced state was found to depend on the upstream activation sequence, GAL4 protein, a positive regulator of transcription, and galactose, the inducing agent. The effect of the GAL4 protein-upstream activation sequence complex on the structure of adjacent chromatin required no other promoter sequences. Although sequences protected by histones in the repressed state became more accessible to micrococcal nuclease and (methidiumpropyl-EDTA)iron(II) cleavage following induction of transcription, DNA-protein particles containing these sequences retained the electrophoretic mobility of nucleosomes, indicating that the promoter region can be associated with nucleosomes under conditions of transcription activation.

ACTIVATION OF YEAST RNA POLYMERASE-II TRANSCRIPTION BY A THYMIDINE-RICH UPSTREAM ELEMENT INVITROPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICALue, N. F., Buchman, A. R., Kornberg, R. D.1989; 86 (2): 486-490

Abstract

A thymidine-rich sequence upstream of the DED1 gene of Saccharomyces cerevisiae activated transcription of the CYC1 promoter by RNA polymerase II in vitro. Activation was inhibited by an excess of an oligonucleotide with the same but not a closely related thymidine-rich sequence, pointing to the involvement of a specific thymidine-rich element-binding factor. The extent of activation was as great as 30-fold and showed a similar distance and orientation dependence and a similar effect of deletions in vitro as in vivo.

Abstract

General regulatory factor I (GRFI) is a yeast protein that binds in vitro to specific DNA sequences at diverse genetic elements. A strategy was pursued to test whether GRFI functions in vivo at the sequences bound by the factor in vitro. Matches to a consensus sequence for GRFI binding were found in a variety of locations: upstream activating sequences (UASs), silencers, telomeres, and transcribed regions. All occurrences of the consensus sequence bound both crude and purified GRFI in vitro. All binding sites for GRFI, regardless of origin, provided UAS function in test plasmids. Also, GRFI binding sites specifically stimulated transcription in a yeast in vitro system, indicating that GRFI can function as a positive transcription factor. The stimulatory effect of GRFI binding sites at UASs for the PYK1 and ENO1 genes is significantly enhanced by flanking DNA elements. By contrast, regulatory sequences that flank the GRFI binding site at HMR E convert this region to a transcriptional silencer.

Abstract

Arrays of nucleosomes were positioned with respect to the GAL1-GAL10 intergenic region inserted into Saccharomyces cerevisiae minichromosomes. Deletions of DNA flanking the upstream activation sequence left the array unaltered, showing that nucleosome positioning was not a consequence of sequence-specific histone-DNA interactions but depended on proximity to the galactose-responsive upstream activation sequence (UASG). Replacement of the upstream activation sequence by synthetic oligonucleotides with different protein-binding properties identified a short sequence within this region that is responsible for the ordered array. This sequence overlaps a binding site for GAL4 protein, a positive regulator of transcription, but exerts its effect on chromatin structure independently of GAL4, probably through binding a novel factor that is not GAL-specific.

Abstract

Escherichia coli RNA polymerase holoenzyme forms two-dimensional crystals when adsorbed to positively charged lipid layers at the air/water interface. Adsorption of the protein is driven by electrostatic interactions between the positively charged lipid surface and the polymerase molecule, which has a net negative charge. Crystallization is dependent on the adsorption and concentration of RNA polymerase on fluid lipid surfaces. Image analysis of electron micrographs of crystals in negative stain, which diffract to 30 A resolution, shows irregularly shaped protein densities about 100 x 160 A, consistent with the dimensions of single polymerase molecules.

Abstract

Expressions are derived for distributions of nucleosomes in chromatin. Nucleosomes are placed on DNA at the densities found in bulk chromatin, and their locations are allowed to vary at random. No further assumptions are required to simulate the periodic patterns of digestion obtained with various nucleases. The introduction of a boundary constraint, due for example to sequence-specific protein binding, results in an array of regularly spaced nucleosomes at nonrandom locations, similar to the arrays reported for some genes and other chromosomal regions.

Abstract

The HMR E silencer is required for SIR-dependent transcriptional repression of the silent mating-type locus, HMR. The silencer also behaves as an origin of replication (ARS element) and allows plasmids to replicate autonomously in yeast. The replication and segregation properties of these plasmids are also dependent on the four SIR genes. We have previously characterized two DNA-binding factors in yeast extracts that recognize specific sequences at the HMR E silencer. These proteins, called ABFI (ARS-Binding Factor) and GRFI (General Regulatory Factor), are not encoded by any of the SIR genes. To investigate the biological roles of these factors, single-base-pair mutations were constructed in both binding sites at the HMR E silencer that were no longer recognized by the corresponding proteins in vitro. Our results indicate that the GRFI-binding site is required for the efficient segregation of plasmids replicated by the HMR E silencer. SIR-dependent transcriptional repression requires either an intact ABFI-binding site or GRFI-binding site, although the GRFI-binding site appears to be more important. A double-mutant silencer that binds neither ABFI nor GRFI does not mediate transcriptional repression of HMR. The replacement of HMR E with a chromosomal origin of replication (ARS1) allows partial SIR-dependent transcriptional repression of HMR, indicating a role for replication in silencer function. Together, these results suggest that the SIR proteins influence the properties of the HMR E silencer through interactions with other DNA-binding proteins.

Abstract

Two-dimensional crystals of cholera toxin bound to receptors in a lipid membrane give diffraction extending to 15 A resolution. Three-dimensional structure determination reveals a ring of five B subunits on the membrane surface, with one-third of the A subunit occupying the center of the ring. The remaining mass of the A subunit appears to penetrate the hydrophobic interior of the membrane. Cleavage of a disulfide bond in the A subunit, which activates the toxin, causes a major conformational change, with the A subunit mostly exiting from the B ring.

Abstract

Two DNA-binding factors from Saccharomyces cerevisiae have been characterized, GRFI (general regulatory factor I) and ABFI (ARS-binding factor I), that recognize specific sequences within diverse genetic elements. GRFI bound to sequences at the negative regulatory elements (silencers) of the silent mating type loci HML E and HMR E and to the upstream activating sequence (UAS) required for transcription of the MAT alpha genes. A putative conserved UAS located at genes involved in translation (RPG box) was also recognized by GRFI. In addition, GRFI bound with high affinity to sequences with the (C1-3A)-repeat region at yeast telomeres. Binding sites for GRFI with the highest affinity appeared to be of the form 5'-(A/G)(A/C)ACCCANNCA(T/C)(T/C)-3', where N is any nucleotide. ABFI-binding sites were located next to autonomously replicating sequences (ARSs) at controlling elements of the silent mating type loci HMR E, HMR I, and HML I and were associated with ARS1, ARS2, and the 2 micron plasmid ARS. Two tandem ABFI binding sites were found between the HIS3 and DED1 genes, several kilobase pairs from any ARS, indicating that ABFI-binding sites are not restricted to ARSs. The sequences recognized by ABFI showed partial dyad-symmetry and appeared to be variations of the consensus 5'-TATCATTNNNNACGA-3'. GRFI and ABFI were both abundant DNA-binding factors and did not appear to be encoded by the SIR genes, whose products are required for repression of the silent mating type loci. Together, these results indicate that both GRFI and ABFI play multiple roles within the cell.

Abstract

The B1 subunit of ribonucleotide reductase formed two-dimensional crystals when bound to and effector nucleotide linked to lipids in planar layers at the air/water interface. The effector lipid consisted of dATP coupled through the gamma-phosphoryl group and an epsilon-aminocaproyl linker to phosphatidylethanolamine. Two-dimensional crystals of B1 reductase, like those of antibodies and cholera toxin obtained previously, formed under physiologic conditions of pH and ionic strength, with no precipitant added to the solution. There was, however, a requirement for dTTP in the solution, presumably to ensure binding of the dATP-lipid at only one of two effector sites on the enzyme. Diffraction from the crystals extended to 18-A resolution in negative stain, with unit cell parameters a = 110 A, b = 277 A, and gamma = 90 degrees. Image analysis revealed the B1 dimer as a pair of roughly cylindrical objects, each 105-109 A in length and 31-34 A in diameter.

ACCURATE INITIATION AT RNA POLYMERASE-II PROMOTERS IN EXTRACTS FROM SACCHAROMYCES-CEREVISIAEPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICALue, N. F., Kornberg, R. D.1987; 84 (24): 8839-8843

Abstract

A yeast nuclear extract supported transcription from the CYC1 and PYK1 promoters. Transcription was initiated in vitro at or near sites used in vivo. Deletion of "TATA" sequences abolished the reaction. alpha-Amanitin (10 micrograms/ml) and chloride (100 mM) were highly inhibitory.

Abstract

The GAL80 protein of Saccharomyces cerevisiae, synthesized in vitro, bound tightly to GAL4 protein and to a GAL4 protein-upstream activation sequence DNA complex, as shown by (i) coimmunoprecipitation of GAL4 and GAL80 proteins with anti-GAL4 antiserum, (ii) an electrophoretic mobility shift of a GAL4 protein-upstream activation sequence DNA complex upon the addition of GAL80 protein, and (iii) GAL4-dependent binding of GAL80 protein to upstream activation sequence DNA immobilized on Sepharose beads. Anti-GAL4 antisera were raised against a GAL4-URA3 fusion protein, which could be purified to homogeneity in a single step with the use of an affinity chromatographic procedure for the URA3 gene product.

Abstract

The poly(A)-binding protein (PAB) gene of Saccharomyces cerevisiae is essential for cell growth. A 66-amino acid polypeptide containing half of a repeated N-terminal domain can replace the entire protein in vivo. Neither an octapeptide sequence conserved among eucaryotic RNA-binding proteins nor the C-terminal domain of PAB is required for function in vivo. A single N-terminal domain is nearly identical to the entire protein in the number of high-affinity sites for poly(A) binding in vitro (one site with an association constant of approximately 2 X 10(7) M-1) and in the size of the binding site (12 A residues). Multiple N-terminal domains afford a mechanism of PAB transfer between poly(A) strands.

Abstract

Promoters were assembled in nucleosomes or ligated to nucleosomes and transcribed with SP6 RNA polymerase or with mammalian RNA polymerase II and accessory factors. Neither polymerase would initiate transcription at a promoter in a nucleosome, but once engaged in transcription, both polymerases were capable of reading through a nucleosome. In the course of readthrough transcription, the histones were displaced from the DNA, as shown by the exposure of restriction sites and by a shift of the template to the position of naked DNA in a gel. It may be true, in general, that processive enzymes will traverse regions of DNA organized in nucleosomes and displace histones.

ISOLATION OF A SACCHAROMYCES-CEREVISIAE CENTROMERE DNA-BINDING PROTEIN, ITS HUMAN HOMOLOG, AND ITS POSSIBLE ROLE AS A TRANSCRIPTION FACTORMOLECULAR AND CELLULAR BIOLOGYBram, R. J., Kornberg, R. D.1987; 7 (1): 403-409

Abstract

A protein that binds specifically to Saccharomyces cerevisiae centromere DNA element I was purified on the basis of a nitrocellulose filter-binding assay. This protein, termed centromere-binding protein 1 (CP1), was heat stable and renaturable from sodium dodecyl sulfate (SDS), and assays of eluates from SDS gels indicated a molecular weight of 57,000 to 64,000. An activity with similar specificity and stability was detected in human lymphocyte extracts, and analysis in SDS gels revealed a molecular weight of 39,000 to 49,000. CP1-binding sites occurred not only at centromeres but also near many transcription units, for example, adjacent to binding sites for the GAL4-positive regulatory protein upstream of the GAL2 gene in S. cerevisiae and adjacent to the TATA element of the adenovirus major late promoter. A factor (termed USF) that binds to the latter site and stimulates transcription has been isolated from HeLa cells by others.

Abstract

The B subunit of cholera toxin forms two-dimensional crystals when bound to its membrane receptor, ganglioside GM1, in phospholipid layers. A rectangular crystal lattice gives diffraction extending to 15-A resolution in negative stain, and image-processing of electron micrographs reveals a ring of five protein densities. The diameter of the central hole and the outer diameter of the ring are about 20 and 60 A, respectively. These data are consistent with a pentameric, doughnut-shaped structure of the B subunit that lies flat on a membrane surface. A hexagonal crystal lattice is obtained as well, and results of image processing and chemical crosslinking allow two interpretations: the B subunit may exist in both pentameric and hexameric forms or, more likely, the hexagonal lattice may represent a disordered or liquid crystalline form, in which a pentamer undergoes rotational averaging about its 5-fold axis.

Abstract

The nuclear envelope defines a compartment boundary which is penetrated by pores that mediate a remarkable transport process. Precursor RNAs are retained in the nucleus, while processed messenger RNA, transfer RNA and ribosomal subunits are transported to the cytoplasm. Proteins destined for the nucleus become localized soon after synthesis and again following mitosis, while cytoplasmic proteins are excluded. The process is highly specific: a single base change in vertebrate initiator tRNAMet (tRNAiMet) reduces the rate of export 20-fold; a point mutation within the simian virus 40 (SV40) large-T antigen, converting Lys 128 to Thr or Asn, prevents import. Lys 128 lies within a short 'signal' sequence which, when fused to large non-nuclear proteins, causes their accumulation in nuclei. Regions of other eukaryotic proteins also seem to contain nuclear localization signals, although a single consensus sequence has not emerged. We report here that a synthetic peptide containing 10 residues of large-T antigen sequence serves as a nuclear localization signal when cross-linked to bovine serum albumin (BSA) or immunoglobulin G (IgG) and microinjected in Xenopus oocytes. Substitution of Thr at the position of Lys 128 in this peptide renders it six- to sevenfold less effective. The uptake of peptide-linked BSA is saturable, and the rate is diminished by co-injection of free peptide. These findings are indicative of a receptor-mediated uptake process. With the use of anti-peptide antibodies, a family of proteins is revealed in nuclear but not cytoplasmic extracts of human lymphocytes which contain large-T antigen-like sequences.

Abstract

Nuclear and cytoplasmic poly(A)-binding proteins have been purified from Saccharomyces cerevisiae, and antisera have been used to isolate a gene that encodes them. The gene occurs in a single copy on chromosome 5 and gives rise to a unique, unspliced 2.1 kb transcript. The nuclear protein appears to be derived from the cytoplasmic one by proteolytic cleavage into 53 and 17 kd polypeptides that remain associated during isolation. DNA sequence determination reveals four tandemly arrayed 90 amino acid regions of homology that probably represent poly(A)-binding domains. A 55 residue A-rich region upstream of the initiator methionine codon in the mRNA shows an affinity for poly(A)-binding protein comparable to that of poly(A)180-220, raising the possibility of feedback regulation of translation.

Abstract

Binding sites for the GAL4-positive regulatory protein have been identified upstream of six galactose-inducible genes of Saccharomyces cerevisiae on the basis of (i) protection in DNAse I footprints, (ii) loss of protection when excess GAL4-binding oligonucleotide is added and (iii) homology with a 23-bp dyad-symmetric consensus sequence. Many of the binding sites have been shown to function as upstream activating sequences. The number of binding sites upstream of the various genes ranges from one to four, but a feature is conserved: in cases of multiple sites there is a pair with highest binding affinity located at dyad--dyad distances of 82--87 bp. We suggest that a pair of sites facilitates repression by the GAL80-negative regulatory protein, on the basis of (i) a correlation of a pair of sites (or only one) with full (or only partial) repression and (ii) the introduction of a second site abolishing transcription occurring with one.

Abstract

Polyadenylate-binding activity can be detected in eluates from sodium dodecyl sulfate gels by a nitrocellulose filter-binding assay. Nuclear extracts from rat liver show a single peak of binding activity at 50 to 55 kilodaltons; cytoplasmic extracts show a single peak at 70 to 80 kilodaltons, corresponding to a 75-kilodalton protein previously described. Similar results are obtained with yeast and mouse fibroblasts, indicating a high degree of conservation of both nuclear and cytoplasmic polyadenylate-binding proteins. The activity from rat liver nuclei has been purified 125-fold on the basis of specific binding to polyadenylate and shows two main bands in sodium dodecyl sulfate gels at 53 and 55 kilodaltons.

SPECIFIC PROTEIN-BINDING TO FAR UPSTREAM ACTIVATING SEQUENCES IN POLYMERASE-II PROMOTERSPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICABram, R. J., Kornberg, R. D.1985; 82 (1): 43-47

Abstract

A binding activity specific for the upstream activating sequence of the GAL1-GAL10 promoter of Saccharomyces cerevisiae has been purified 220-fold on the basis of a nitrocellulose filter-binding assay. The binding activity is enriched in a nuclear preparation and is likely to be the GAL4 gene product. DNase I-protection mapping patterns reveal binding to two 30-base-pair regions at the boundaries of the sequence. A nearly identical mapping pattern is obtained with the coordinately regulated GAL7 promoter. The four 30-base-pair regions of binding in the two promoters are closely homologous, with a core consensus sequence of C-G-CG-TG-C-A-A-C-A-G-T-G-C-T-C-C-G-A-A- GC-G-A-T. A synthetic oligonucleotide with such a sequence competes with the upstream activating sequence in the binding reaction.

Abstract

Two methods have been used to measure the single-strand lengths of the DNA fragments produced by deoxyribonuclease I digestion of chromatin. The average lengths obtained are muliples of about 10.4 bases, significantly different from the value of 10 previously reported. This periodicity in fragment lengths is closely related to the periodicity of the DNA double helix in chromatin, but the two values need not be exactly the same.

RELATION OF NUCLEOSOMES TO NUCLEOTIDE-SEQUENCES IN RATPHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY OF LONDON SERIES B-BIOLOGICAL SCIENCESPrunell, A., Kornberg, R. D.1978; 283 (997): 269-273

Abstract

The relation of nucleosomes to nucleotide sequences is random for most single copy sequences in rat liver. This could be due to variation in the DNA content of nucleosomes, and a procedure for detecting such variation is described.